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

  • Mark Hilge
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University Basel, CH-4058 Basel, Switzerland
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  • Author Footnotes
    * This work was supported by grants from the Swiss National Science Foundation (National Centres of Competence for Research (NCCRs) Structural Biology and TransCure) and the Swiss Initiative for Systems Biology (SystemsX.ch). This article is part of the Thematic Minireview Series on Ins and Outs of Calcium Transport.
    This article contains supplemental Fig. S1.
Open AccessPublished:July 20, 2012DOI:https://doi.org/10.1074/jbc.R112.353573
      The binding of Ca2+ to two adjacent Ca2+-binding domains, CBD1 and CBD2, regulates ion transport in the Na+/Ca2+ exchanger. As sensors for intracellular Ca2+, the CBDs form electrostatic switches that induce the conformational changes required to initiate and sustain Na+/Ca2+ exchange. Depending on the presence of a few key residues in the Ca2+-binding sites, zero to four Ca2+ ions can bind with affinities between 0.1 to 20 μm. Importantly, variability in CBD2 as a consequence of alternative splicing modulates not only the number and affinities of the Ca2+-binding sites in CBD2 but also the Ca2+ affinities in CBD1.

      Introduction

      In the heart, the Na+/Ca2+ exchanger (NCX)
      The abbreviations used are: NCX
      Na+/Ca2+ exchanger
      CBD
      Ca2+-binding domain
      PDB
      Protein Data Bank
      CLD
      catenin-like domain
      SAXS
      small-angle x-ray scattering
      mGluR
      metabotropic glutamate receptor
      PIP2
      phosphatidylinositol 4,5-bisphosphate.
      works in concert with the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases to remove Ca2+ released into the cytosol after the muscle contraction phase (Refs.
      • Blaustein M.P.
      • Lederer W.J.
      Sodium/calcium exchange: its physiological implications.
      ,
      • Philipson K.D.
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      Sodium/calcium exchange: a molecular perspective.
      ,
      • Annunziato L.
      • Pignataro G.
      • Di Renzo G.F.
      Pharmacology of brain Na+/Ca2+ exchanger: from molecular biology to therapeutic perspectives.
      ,
      • Lytton J.
      Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport.
      ,
      • Brini M.
      • Carafoli E.
      The plasma membrane Ca2+-ATPase and the plasma membrane sodium/calcium exchanger cooperate in the regulation of cell calcium.
      ; for general reviews, see Refs.
      • Carafoli E.
      Calcium signaling: a tale for all seasons.
      ,
      • Bers D.M.
      Cardiac excitation-contraction coupling.
      ,
      • Berridge M.J.
      • Bootman M.D.
      • Roderick H.L.
      Calcium signaling: dynamics, homeostasis, and remodeling.
      ). In this process, NCX predominantly exports one Ca2+ ion for the uptake of three Na+ ions (
      • Kang T.M.
      • Hilgemann D.W.
      Multiple transport modes of the cardiac Na+/Ca2+ exchanger.
      ) at an estimated turnover rate of 2500–5000 s−1 (
      • Hilgemann D.W.
      • Nicoll D.A.
      • Philipson K.D.
      Charge movement during Na+ translocation by native and cloned cardiac Na+/Ca2+ exchanger.
      ,
      • Niggli E.
      • Lederer W.J.
      Molecular operations of the sodium/calcium exchanger revealed by conformation currents.
      ,
      • Egger M.
      • Niggli E.
      Regulatory function of sodium/calcium exchange in the heart: milestones and outlook.
      ).
      NCX exists in three isoforms, NCX1 (
      • Nicoll D.A.
      • Longoni S.
      • Philipson K.D.
      Molecular cloning and functional expression of the cardiac sarcolemmal Na+/Ca2+ exchanger.
      ), NCX2 (
      • Li Z.
      • Matsuoka S.
      • Hryshko L.V.
      • Nicoll D.A.
      • Bersohn M.M.
      • Burke E.P.
      • Lifton R.P.
      • Philipson K.D.
      Cloning of the NCX2 isoform of the plasma membrane Na+/Ca2+ exchanger.
      ), and NCX3 (
      • Nicoll D.A.
      • Quednau B.D.
      • Qui Z.
      • Xia Y.R.
      • Lusis A.J.
      • Philipson K.D.
      Cloning of a third mammalian Na+/Ca2+ exchanger, NCX3.
      ). Numerous splice variants have been described for NCX1 (
      • Kofuji P.
      • Lederer W.J.
      • Schulze D.H.
      Mutually exclusive and cassette exons underlie alternatively spliced isoforms of the sodium/calcium exchanger.
      ,
      • Lee S.L.
      • Yu A.S.
      • Lytton J.
      Tissue-specific expression of Na+/Ca2+ exchanger isoforms.
      ) and NCX3 (
      • Quednau B.D.
      • Nicoll D.A.
      • Philipson K.D.
      Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat.
      ). In NCX1, these are encoded by one of the mutually exclusive exons, A or B, in combination with small cassette exons C–F. In this pool of splice forms, the AD variant (detected in the brain) constitutes the shortest form, and the ACDEF form (found in the heart) constitutes the longest splice variant. Based on the presence of two conserved regions in the transmembrane domain, generally referred to as α-repeats (
      • Nicoll D.A.
      • Hryshko L.V.
      • Matsuoka S.
      • Frank J.S.
      • Philipson K.D.
      Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+/Ca2+ exchanger.
      ), NCX has been assigned to the cation/Ca2+ exchanger superfamily (
      • Cai X.
      • Lytton J.
      The cation/Ca2+ exchanger superfamily: phylogenetic analysis and structural implications.
      ). Probably originating from an ancient gene duplication event (
      • Nicoll D.A.
      • Hryshko L.V.
      • Matsuoka S.
      • Frank J.S.
      • Philipson K.D.
      Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+/Ca2+ exchanger.
      ,
      • Rapp M.
      • Granseth E.
      • Seppälä S.
      • von Heijne G.
      Identification and evolution of dual-topology membrane proteins.
      ,
      • Hilge M.
      • Aelen J.
      • Perrakis A.
      • Vuister G.W.
      Structural basis for Ca2+ regulation in the Na+/Ca2+ exchanger.
      ), these α-repeat regions were until recently believed to form oppositely oriented re-entrant loops that were suggested to be involved in ion binding and translocation (
      • Nicoll D.A.
      • Hryshko L.V.
      • Matsuoka S.
      • Frank J.S.
      • Philipson K.D.
      Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+/Ca2+ exchanger.
      ,
      • Iwamoto T.
      • Uehara A.
      • Imanaga I.
      • Shigekawa M.
      The Na+/Ca2+ exchanger NCX1 has oppositely oriented reentrant loop domains that contain conserved aspartic acids whose mutation alters its apparent Ca2+ affinity.
      ).
      Over two decades ago (
      • DiPolo R.
      Calcium influx in internally dialyzed squid giant axons.
      ,
      • Hilgemann D.W.
      Regulation and deregulation of cardiac Na+/Ca2+ exchange in giant excised sarcolemmal membrane patches.
      ) it was demonstrated that NCX not only transports Ca2+ and Na+ but is also regulated by these ions. In pioneering work, Hilgemann et al. (
      • Hilgemann D.W.
      • Collins A.
      • Matsuoka S.
      Steady-state and dynamic properties of cardiac sodium/calcium exchange. Secondary modulation by cytoplasmic calcium and ATP.
      ,
      • Hilgemann D.W.
      • Matsuoka S.
      • Nagel G.A.
      • Collins A.
      Steady-state and dynamic properties of cardiac sodium/calcium exchange. Sodium-dependent inactivation.
      ) dissected the regulatory properties into Ca2+-dependent activation and Na+-dependent inactivation. While initial activation occurs at submicromolar Ca2+ concentrations, counteracting Na+-dependent exchanger inactivation may establish when Na+ concentrations rise above 15 mm. Strikingly, NCX1 splice variants encoded by exon A can alleviate Na+-dependent inactivation at elevated intracellular Ca2+ concentrations, whereas those variants encoded by exon B (
      • Dyck C.
      • Omelchenko A.
      • Elias C.L.
      • Quednau B.D.
      • Philipson K.D.
      • Hnatowich M.
      • Hryshko L.V.
      Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na+/Ca2+ exchanger.
      ,
      • Dunn J.
      • Elias C.L.
      • Le H.D.
      • Omelchenko A.
      • Hryshko L.V.
      • Lytton J.
      The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms.
      ,
      • Hurtado C.
      • Prociuk M.
      • Maddaford T.G.
      • Dibrov E.
      • Mesaeli N.
      • Hryshko L.V.
      • Pierce G.N.
      Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload.
      ) cannot. Furthermore, it appears that exon A-encoded exchanger variants are expressed predominantly in excitable cells, where high Ca2+ fluxes are needed (
      • Qiu Z.
      • Nicoll D.A.
      • Philipson K.D.
      Helix packing of functionally important regions of the cardiac Na+/Ca2+ exchanger.
      ), whereas exon B-encoded variants are generally restricted to non-excitable cells (
      • Quednau B.D.
      • Nicoll D.A.
      • Philipson K.D.
      Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat.
      ).

      Structural Basis for Ca2+ Regulation in NCX

      Structurally, NCX consists of an α-helical transmembrane domain and a large ∼500-residue-long cytosolic loop (Fig. 1A) (
      • Iwamoto T.
      • Nakamura T.Y.
      • Pan Y.
      • Uehara A.
      • Imanaga I.
      • Shigekawa M.
      Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.
      ,
      • Nicoll D.A.
      • Ottolia M.
      • Lu L.
      • Lu Y.
      • Philipson K.D.
      A new topological model of the cardiac sarcolemmal Na+/Ca2+ exchanger.
      ). Residues 371–509 within this loop have been shown to bind Ca2+ with high affinity (Kd ∼ 140–400 nm) (
      • Levitsky D.O.
      • Nicoll D.A.
      • Philipson K.D.
      Identification of the high affinity Ca2+-binding domain of the cardiac Na+/Ca2+ exchanger.
      ,
      • Ottolia M.
      • Philipson K.D.
      • John S.
      Conformational changes of the Ca2+ regulatory site of the Na+/Ca2+ exchanger detected by FRET.
      ). The solution structure of residues 371–509 of canine NCX1 (
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ) redefined the boundaries (Fig. 1B) of a module earlier described as the Calx-β domain (
      • Schwarz E.M.
      • Benzer S.
      Calx, a sodium/calcium exchanger gene of Drosophila melanogaster.
      ). A single copy of the Calx-β domain is found in integrin β4 (
      • Schwarz E.M.
      • Benzer S.
      Calx, a sodium/calcium exchanger gene of Drosophila melanogaster.
      ,
      • Alonso-García N.
      • Inglés-Prieto A.
      • Sonnenberg A.
      • de Pereda J.M.
      Structure of the Calx-β domain of the integrin β4 subunit: insights into function and cation-independent stability.
      ), and multiple copies are present in very large G protein-coupled receptors (
      • Nikkila H.
      • McMillan D.R.
      • Nunez B.S.
      • Pascoe L.
      • Curnow K.M.
      • White P.C.
      Sequence similarities between a novel putative G protein-coupled receptor and Na+/Ca2+ exchangers define a cation-binding domain.
      ,
      • McMillan D.R.
      • Kayes-Wandover K.M.
      • Richardson J.A.
      • White P.C.
      Very large G protein-coupled receptor-1, the largest known cell surface protein, is highly expressed in the developing central nervous system.
      ) and extracellular matrix proteins ranging from sponges to humans (
      • Fernàndez-Busquets X.
      • Kammerer R.A.
      • Burger M.M.
      A 35-kDa protein is the basic unit of the core from the 2 × 104-kDa aggregation factor responsible for species-specific cell adhesion in the marine sponge Microciona prolifera.
      ,
      • Hodor P.G.
      • Illies M.R.
      • Broadley S.
      • Ettensohn C.A.
      Cell-substrate interactions during sea urchin gastrulation: migrating primary mesenchyme cells interact with and align extracellular matrix fibers that contain ECM3, a molecule with NG2-like and multiple calcium-binding domains.
      ). A sequence identity of 27% for residues 501–650 with residues 371–500 suggested the presence of an unexpected, second Ca2+-binding domain (CBD) in the cytosolic exchanger loop (
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ). As for the first, the NMR structure of the second CBD was determined in the Ca2+-bound form. Both domains (CBD1 and CBD2) have the architecture of a β-sandwich, formed by two antiparallel β-sheets, with one β-sheet containing strands A, B, and E and the other containing strands C, D, F, and G (Fig. 1B). Each domain features a β-bulge in strand A, but only CBD2 displays a second β-bulge in strand G. In contrast, CBD1 possesses a cis-proline at a structurally similar position. The long FG loop in each of the individual domains (residues 468–482 in CBD1 and residues 599–627 in CBD2) is largely unstructured. A variable region comprising 39–75 residues, depending on the splice form, precedes the FG loop in CBD2. Superposition of the ensemble of NMR structures of CBD1 and CBD2 (supplemental Fig. S1) shows the largest differences to be in the BC, CD, and FG loops as well as in parts of the first acidic segment (residues 446–454 and 577–582, respectively). Ile-374/Phe-505, Phe-376/Phe-507, Tyr-422/Phe-553, Phe-431/Phe-562, Phe-456/Phe-587, and Leu-460/Ile-591 form the core of the two domains and are probably important for stability. Intriguingly, at the N-terminal side of the domains, the highly conserved Arg-396/Arg-527 (Fig. 1B) engages in a stacking interaction with the conserved Phe-431/Phe-562. Whereas Arg-527 in CBD2 clearly stabilizes the strictly conserved BC loop, the function of Arg-396 in CBD1 remains unclear.
      Figure thumbnail gr1
      FIGURE 1Structural basis and organization of the two Ca2+ sensors (CBD1 and CBD2) in NCX. A, updated topology model of NCX consisting of a transmembrane domain and an ∼500-residue-long cytosolic loop that harbors the two Ca2+-binding domains (CBD1 and CBD2). The variable region in CBD2 as a consequence of alternative splicing (AS) is shown in violet. The conserved α-repeat regions that define the cation/Ca2+ exchanger superfamily are colored yellow. B, ribbon diagram displaying the β-sandwich architecture of Ca2+-bound CBD1. The structures of the CBDs redefined the previously described Calx-β motif by adding strands A and G (yellow). C and D, detailed views of the CBD1 (PDB code 2DPK) and CBD2 (code 2QVM) Ca2+-binding sites in the Ca2+-bound form. E, hypothetical model of NCX consisting of four domains: transmembrane domain (residues 1–217 and 727–903; gray), the CLD (residues 218–370 and 651–726; blue), CBD1 (residues 371–500; red), and CBD2 (residues 501–650; green), with the numbering based on the canine NCX1 AD splice variant (NCX1.4). The ribbon diagram of CBD2 (upper right) displays the variable region encoded by the mutually exclusive exons A or B (blue) and the small cassette exons (yellow) at the opposite side of the CBD2 Ca2+-binding sites. F, interface of CBD1 and CBD2, displaying the crucial salt bridges and hydrogen bonds in the presence of Ca2+. Residues in parentheses refer to the numbering scheme of canine NCX1.
      Subsequent x-ray structures of Ca2+-bound CBDs (CBD1, Protein Data Bank (PDB) code 2DPK (
      • Nicoll D.A.
      • Sawaya M.R.
      • Kwon S.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+-binding motif.
      ) and codes 3E9T and 3EAD (
      • Wu M.
      • Le H.D.
      • Wang M.
      • Yurkov V.
      • Omelchenko A.
      • Hnatowich M.
      • Nix J.
      • Hryshko L.V.
      • Zheng L.
      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.
      ); and CBD2, code 2QVM (
      • Besserer G.M.
      • Ottolia M.
      • Nicoll D.A.
      • Chaptal V.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
      )) have provided a more detailed view of the Ca2+-binding sites. The structures show that the largest contribution to the Ca2+-binding sites of the CBDs originates from two acidic segments, located in the EF loop and at the C terminus of the domains (Fig. 1, B–D), respectively. Interestingly, in CBD2, the EF loop is encoded by one of the mutually exclusive exons, A or B, which determines whether NCX1 can overcome Na+-dependent inactivation. In both CBDs, Ca2+-binding sites are complemented by conserved glutamate (Glu-385/Glu-516) and aspartate (Asp-421/Asp-552) residues in the AB and CD loops, respectively. In CBD1, four Ca2+ ions are bound in an arrangement reminiscent of C2 domains (Fig. 1C) (
      • Nicoll D.A.
      • Sawaya M.R.
      • Kwon S.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+-binding motif.
      ). In contrast, due to Lys-585 and the absence of acidic counterparts of Glu-454, Asp-499, and Asp-500 of CBD1, the Ca2+-binding sites of CBD2-AD (Fig. 1D), with AD denoting the presence of residues encoded by exons A and D, show a reduced density of negative charges. Hence, CBD2-AD coordinates only two Ca2+ ions (
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ,
      • Besserer G.M.
      • Ottolia M.
      • Nicoll D.A.
      • Chaptal V.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
      ), whereas Arg-578 and Cys-585 in exon B-encoded NCX1 splice variants prevent binding of Ca2+ completely (
      • Dunn J.
      • Elias C.L.
      • Le H.D.
      • Omelchenko A.
      • Hryshko L.V.
      • Lytton J.
      The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms.
      ,
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ).
      Comparison of 15N heteronuclear single quantum coherence NMR spectra of the Ca2+-free and Ca2+-bound forms suggested that there is a substantial loss of structural integrity in the Ca2+-binding sites of CBD1 in the absence of Ca2+, but only a modest degree of disorder in the Ca2+-binding sites of CBD2-AD (
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ). These preliminary indications were later confirmed by an x-ray structure of the Ca2+-free form of CBD1 (PDB code 3E9T, chains C and D) (
      • Wu M.
      • Le H.D.
      • Wang M.
      • Yurkov V.
      • Omelchenko A.
      • Hnatowich M.
      • Nix J.
      • Hryshko L.V.
      • Zheng L.
      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.
      ), where no electron density was visible for residues corresponding to Asp-447–Glu-451 and Asp-498–Asp-500. In the case of CBD2-AD, the NMR structure of the apo-form (PDB code 2KLS) (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ) shows that Lys-585 crucially points into Ca2+-binding site II, where it forms a salt bridge with Asp-552 and thereby stabilizes the Ca2+-binding sites. Intriguingly, this NMR structure, determined at neutral pH, does not reflect a state as rigid and locked as the state suggested by an x-ray structure of supposedly Ca2+-free CBD2-AD (PDB code 2QVK) (
      • Besserer G.M.
      • Ottolia M.
      • Nicoll D.A.
      • Chaptal V.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
      ) obtained from crystals grown at pH 4.9.
      Using the CBDs as constraints resulted in a model for the intact exchanger (Fig. 1E) in which the Ca2+-binding sites of CBD1 are ∼90 Å away from the transport Ca2+-binding site in the transmembrane domain (
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ). In contrast, the Ca2+-binding sites of CBD2 are close to a predicted third domain that shows homology to α-catenin (catenin-like domain (CLD)). The predicted oppositely oriented arrangement of the CBDs was subsequently confirmed by solution structures obtained from small-angle x-ray scattering (SAXS) (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ) and by x-ray structures of CBD12 from Drosophila isoforms CalX1.1 and CalX1.2 (
      • Wu M.
      • Tong S.
      • Gonzalez J.
      • Jayaraman V.
      • Spudich J.L.
      • Zheng L.
      Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing.
      ), two-domain constructs containing CBD1 and CBD2.
      In CalX1.1, the interface of CBD1 and CBD2 displays a relative paucity of interactions between the domains (Fig. 1F). Most importantly, Arg-584, the residue corresponding to the strictly conserved Arg-532 of canine NCX1, forms a strong salt bridge with Asp-552 (Asp-500) as well as two hydrogen bonds with Asp-517 (Asp-448) and Asp-552 (Asp-500), respectively. In addition, Arg-584 (Arg-532) is further stabilized via a hydrogen bond to Asn-615 (Asn-564). In the center of the interface, the side chain of His-553 (His-501) forms two hydrogen bonds with the backbone carbonyls of Leu-677 and Lys-679. At the opposite side of the interface, Glu-521 (Glu-452) maintains a bidentate salt bridge with the conserved Arg-673, located in helix 2 of CBD2. Finally, Phe-519 (Phe-450) at the tip of the EF loop may be involved in some limited van der Waals interactions with the side chains of His-553, Ile-674, and Ser-678. While the interface explains previously intriguing functions of the strictly conserved Asp-517 (Asp-448) (
      • Matsuoka S.
      • Nicoll D.A.
      • Hryshko L.V.
      • Levitsky D.O.
      • Weiss J.N.
      • Philipson K.D.
      Regulation of the cardiac Na+/Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+-binding domain.
      ) and Glu-521 (Glu-452), strikingly, the apparent structural importance of Arg-584 (Arg-532) in the CBD12 interface (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ,
      • Wu M.
      • Tong S.
      • Gonzalez J.
      • Jayaraman V.
      • Spudich J.L.
      • Zheng L.
      Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing.
      ) is not in line with functional data for this residue (
      • Ottolia M.
      • Nicoll D.A.
      • John S.
      • Philipson K.D.
      Interactions between Ca2+-binding domains of the Na+/Ca2+ exchanger and secondary regulation.
      ).
      A recent major development in our understanding of Na+/Ca2+ exchange was the determination of the structure of a prokaryotic NCX homolog from Methanococcus jannaschii (NCX_Mj) in the outward conformation (
      • Liao J.
      • Li H.
      • Zeng W.
      • Sauer D.B.
      • Belmares R.
      • Jiang Y.
      Structural insight into the ion exchange mechanism of the sodium/calcium exchanger.
      ). The 1.9 Å crystal structure revealed a largely symmetrical transmembrane domain, arranged in the form of two structurally similar halves of five α-helices with opposite topology (Fig. 2A). Helices 2–5 and 7–10 are tightly packed, whereas helices 1 and 6 are somewhat separated from the bundle and tilted at an angle of ∼45° with respect to the vertical axis of the membrane. In line with the proposed transport stoichiometry of one Ca2+ ion in exchange for three Na+ ions, four cation-binding sites, one specific for Ca2+ and three that probably bind Na+, were identified in the protein core at the center of the membrane. Strikingly, all residues participating in ion binding originate from the α-repeat regions and are highly conserved in the NCX and Na+/Ca2+-K+ exchanger branches of the cation/Ca2+ exchanger superfamily (
      • Cai X.
      • Lytton J.
      The cation/Ca2+ exchanger superfamily: phylogenetic analysis and structural implications.
      ). In particular, in the center, Glu-54 (Glu-113 in canine NCX1) and Glu-213 (Asp-814) coordinate the Ca2+ ion via their carboxylate groups, whereas the backbone carbonyl oxygen atoms of Thr-50 and Thr-209 complement the binding site. In addition, the Oϵ2 atoms of Glu-54 and Glu-213 contribute to the middle Na+-binding site as well as to the Na+-binding sites toward the extra- and intracellular sides, respectively. The latter two Na+-binding sites share identical ligand chemistry and geometry, where Ser-77, Ala-206, Thr-209, and Ser-210 form the Na+-binding site oriented toward the extracellular side, and Ala-47, Thr-50, Ser-51, and Ser-236 constitute the Na+-binding site closer to the intracellular side. Finally, Asn-81 and Asp-240 complete the coordination scheme of the middle Na+-binding site.
      Figure thumbnail gr2
      FIGURE 2Structure of the NCX homolog from M. jannaschii and a hypothetical model for intact NCX1. A, ribbon diagram displaying symmetry-related helices 1–5 (light gray) and 6–10 (dark gray). The inset shows the detailed coordination of three Na+ ions and one Ca2+ ion. B, summary of the available structural data assembled in a hypothetical model for intact NCX1. Residues for which no structural data are available are indicated. The orientation of the domains with respect to each other is arbitrary, as the linker regions between the CLD and the transmembrane domain, as well as the CBDs, are missing. The model for the transmembrane domain of NCX1 is based on the structure of NCX_Mj (PDB code 3V5U), whereas the homology model of the CLD is derived from the structure of α-catenin (code 1H6G).
      Two hydrophilic pathways permit independent access to the Na+- and Ca2+-binding sites from the extracellular side. Making use of the symmetry of NCX_Mj by applying a molecular dyad defined by the middle Na+ ion and the Ca2+ ion also enabled the construction of a feasible model for the inward-facing conformation that reflects the access pathways from the cytosolic side. The crystal structure of the outward-facing conformation of NCX_Mj and the inward-facing model led to the proposal of a simple and rapid Na+/Ca2+ exchange mechanism that is in agreement with the high turnover rates found for NCX. Together, this work has provided another substantial piece of information to help solve the puzzle of the intact structure of mammalian NCX (Fig. 2B). Presently, residues 1–42, 218–258, and 651–726 (numbering according to the AD splice variant of NCX1), which determine the relative positions of the domains with respect to each other, are the remaining missing pieces.

      Ca2+ Binding Determinants of CBD1 and CBD2

      The thermodynamic properties of the Ca2+-binding sites of CBD1 and CBD2 have been extensively investigated by isothermal titration calorimetry (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ). Initial sequence comparison of all available Na+/Ca2+ exchanger sequences revealed strict conservation of the Ca2+-coordinating residues in CBD1 (Fig. 1C). The expected similarity of the Ca2+ binding properties of the three known exchanger isoforms was confirmed by the titration of CBD1 of canine NCX1 and mouse NCX2 and NCX3 (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ). However, these experiments did not allow their individual macroscopic binding constants to be confidently determined, but only the approximate range, 100–600 nm. These affinities make CBD1 the primary Ca2+ sensor and thus responsible for Ca2+ activation in NCX (
      • Hilgemann D.W.
      • Collins A.
      • Matsuoka S.
      Steady-state and dynamic properties of cardiac sodium/calcium exchange. Secondary modulation by cytoplasmic calcium and ATP.
      ,
      • Levitsky D.O.
      • Nicoll D.A.
      • Philipson K.D.
      Identification of the high affinity Ca2+-binding domain of the cardiac Na+/Ca2+ exchanger.
      ,
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ).
      In strong contrast to CBD1, sequence analysis of CBD2 has revealed that residue types at positions 552, 578, and 585, located around Ca2+-binding site II (Fig. 1D), can vary depending on the isoform and splice variant. As a consequence, the different isoforms and splice variants bind zero to three Ca2+ ions with affinities ∼5–50-times lower than those of CBD1 (for details, see Ref.
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ).

      Function of Residues Encoded by the Cassette Exons

      An increasing amount of evidence indicates that besides the Ca2+-binding sites of CBD1 and CBD2, residues encoded by the cassette exons may contribute to ion transport regulation in NCX. The first sign that cassette exons can influence Ca2+ affinities of the CBD1 Ca2+-binding sites came from isothermal titration calorimetry experiments (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ) with CBD12-AD and CBD12-ACDEF, two-domain constructs representing the brain and heart splice variants. Although the individual CBD structures suggest that both constructs should bind six Ca2+ ions, strikingly, the longer ACDEF variant appears to bind only two Ca2+ ions with medium affinity, whereas CBD12-AD binds six Ca2+ ions, as expected. Completely lacking the high affinity component of CBD1, particle-induced x-ray emission experiments (
      • Garman E.F.
      • Grime G.W.
      Elemental analysis of proteins by microPIXE.
      ) confirmed the coordination of four Ca2+ ions in CBD12-ACDEF prior to the start of titration. Thus, residues encoded by the additional cassette exons C, E, and F, located at the opposite end of the CBD2 Ca2+-binding sites, apparently increase the CBD1 Ca2+ affinities substantially. This is in line with the fact that the ACDEF splice variant is found predominantly in the heart, where the exchanger either is required to enter the activated state at considerably lower intracellular Ca2+ concentrations or is even permanently activated (
      • Reeves J.P.
      • Condrescu M.
      Ionic regulation of the cardiac sodium/calcium exchanger.
      ) to guarantee high Ca2+ fluxes.

      Electrostatic Switches in CBD1 and CBD2

      In analogy to C2 domains (
      • Shao X.
      • Li C.
      • Fernandez I.
      • Zhang X.
      • Südhof T.C.
      • Rizo J.
      Synaptotagmin-syntaxin interaction: the C2 domain as a Ca2+-dependent electrostatic switch.
      ), analysis of the electrostatic potentials of CBD1 and CBD2 in the absence and presence of Ca2+ revealed dramatic differences and suggested that the CBD1 and CBD2 Ca2+-binding sites form electrostatic switches. In particular, in the absence of Ca2+, the CBD1 Ca2+-binding sites display a strongly negative electrostatic potential due to the extensive cluster of aspartic and glutamic acid residues (Asp-421, Asp-446, Asp-447, Asp-448, Asp-498, Asp-499, Asp-500, Glu-385, Glu-451, and Glu-454) and the lack of any basic residues (Fig. 1C). This high density of negative charges not only is responsible for the loss of the structural integrity of the CBD1 Ca2+-binding sites in the absence of Ca2+ but may also cause repulsion with a highly acidic conserved region consisting of four consecutive glutamate residues (Glu-622–Glu-625) at the beginning of helix 2 in CBD2. Upon binding of four Ca2+ ions, both the negative potential and the repulsion are likely to be strongly reduced.
      In comparison, the CBD2 electrostatic potential in the absence of Ca2+ is considerably less negative due to the lower number of acidic residues and the presence of basic residues (Arg-547, Lys-583, and Lys-585) around Ca2+-binding site II. In addition, the different numbers and affinities of the Ca2+-binding sites in the various isoforms and splice variants determine the strength of the electrostatic switch in CBD2.

      Ca2+ Induces a Twist in the Hinge between CBD1 and CBD2

      SAXS analysis of CBD12-AD and CBD12-ACDEF constructs in the absence and presence of Ca2+ revealed a substantial conformational change upon Ca2+ binding that leads to a compaction of the CBDs (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ). Although in the intact exchanger this conformational change is probably constrained by the N- and C-terminal linker regions to the CLD, the SAXS analysis provided the first clue of the effects of Ca2+ binding to the CBD1 Ca2+-binding sites and indicated that the conformational change induced may not be identical in CBD12-AD and CBD12-ACDEF. Indeed, superposition of the crystal structures of CBD12 from the Drosophila Na+/Ca2+ exchanger isoforms CalX1.1 and CalX1.2 shows a rotation by ∼9° between them. Comparison of the interfaces of CalX1.1 and CalX1.2 reveals that the number of interactions between CBD1 and CBD2 is reduced in CalX1.2 with respect to CalX1.1. Most prominently, CalX1.2 apparently lacks the salt bridge between Glu-521 and Arg-673 (Fig. 1F), resulting in increased interdomain flexibility.

      The Drosophila Exchanger Remains Enigmatic

      CalX is unique among the characterized NCXs in that micromolar levels of Ca2+ inhibit rather than activate Na+/Ca2+ exchange (
      • Hryshko L.V.
      • Matsuoka S.
      • Nicoll D.A.
      • Weiss J.N.
      • Schwarz E.M.
      • Benzer S.
      • Philipson K.D.
      Anomalous regulation of the Drosophila Na+/Ca2+ exchanger by Ca2+.
      ). With respect to the CBDs, there are at least three major differences between CalX and the other NCXs. First, CBD1 of CalX lacks most of the FG loop that is disordered in the isolated CBD1 structure of canine NCX1 (
      • Hilge M.
      • Aelen J.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.
      ,
      • Nicoll D.A.
      • Sawaya M.R.
      • Kwon S.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+-binding motif.
      ). Similar to the FG loop of CBD2 at the CBD12 interface, the FG loop of CBD1 may rigidify in intact NCX and potentially interact with the CLD. Indeed, Hryshko and co-workers (
      • Dyck C.
      • Maxwell K.
      • Buchko J.
      • Trac M.
      • Omelchenko A.
      • Hnatowich M.
      • Hryshko L.V.
      Structure-function analysis of CALX1.1, a Na+/Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.
      ) could reinstall normal Ca2+ activation in a CalX chimera that had Lys-404–Tyr-580 substituted with the corresponding residues of NCX1, indicating that the trimmed FG loop in CBD1 might play a role in Ca2+ inhibition of CalX. Second, CalX lacks three of the four conserved consecutive glutamate residues that might cause repulsion with the CBD1 Ca2+-binding sites in the absence of Ca2+. Third, CalX does not show Ca2+ binding to CBD2 (PDB code 3E9U (
      • Wu M.
      • Wang M.
      • Nix J.
      • Hryshko L.V.
      • Zheng L.
      Crystal structure of CBD2 from the Drosophila Na+/Ca2+ exchanger: diversity of Ca2+ regulation and its alternative splicing modification.
      )) and is therefore incapable of forming an electrostatic switch in the second CBD. However, unlike in the exon B-encoded variants of NCX1, the region around the EF loop of the individual CBD2 of CalX is structured, although the same region is intriguingly disordered in the Ca2+-bound CBD12 structures (
      • Wu M.
      • Tong S.
      • Gonzalez J.
      • Jayaraman V.
      • Spudich J.L.
      • Zheng L.
      Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing.
      ).

      Potential Mechanism

      The combination of available structural and biophysical data with results from functional studies has resulted in the proposal of a dual electrostatic switch mechanism for Ca2+ regulation in NCX (Fig. 3, A–C) (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ). As intracellular Ca2+ concentrations rise, Ca2+ ions initially bind to the CBD1 Ca2+-binding sites. Increased rigidity and a substantial increase in the electrostatic potential of the Ca2+-binding sites thereby induce a conformational change that leads to a compaction of the CBDs as visualized by SAXS analysis and supported by FRET studies using full-length NCX and CBD12 constructs (
      • John S.A.
      • Ribalet B.
      • Weiss J.N.
      • Philipson K.D.
      • Ottolia M.
      Ca2+-dependent structural rearrangements within Na+/Ca2+ exchanger dimers.
      ). As a consequence, the tension on the N- and/or C-terminal linker region to the CLD that relays Ca2+ binding and release events to the transmembrane domain is most likely reduced. This initial activation step (Fig. 3B) is referred to as Ca2+-dependent activation and initiates Na+/Ca2+ exchange. Demonstrated by particle-induced x-ray emission analysis (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ) and suggested by FRET measurements (
      • John S.A.
      • Ribalet B.
      • Weiss J.N.
      • Philipson K.D.
      • Ottolia M.
      Ca2+-dependent structural rearrangements within Na+/Ca2+ exchanger dimers.
      ), the Ca2+ concentration at which NCX becomes activated probably depends on the residues encoded by the cassette exons that modulate the affinities of the CBD1 Ca2+-binding sites. This modulation would, for instance, allow the appropriate response to single Ca2+ transients produced by type 1 metabotropic glutamate receptors (mGluR1) at Purkinje cell synapses, whereas in hippocampal neurons, mGluR5 generates a radically different Ca2+ signal in the form of an oscillatory pattern (
      • Berridge M.J.
      • Bootman M.D.
      • Roderick H.L.
      Calcium signaling: dynamics, homeostasis, and remodeling.
      ). Although there are no cell-specific expression data for exchanger splice variants in the brain, coexpression of NCX1.4 (encoded by exons A and D) with mGluR1 and of NCX1.5 (encoded by exons A, D, and F) with mGluR5 would make sense, as the latter probably features higher Ca2+ affinities at the CBD1 Ca2+-binding sites and thereby could remain activated during the series of action potentials.
      Figure thumbnail gr3
      FIGURE 3Hypothetical dual electrostatic switch mechanism of Ca2+ regulation in NCX. A, inactive Ca2+-free NCX in extended conformation. B, submicromolar Ca2+ concentrations induce a conformational change via the electrostatic switch in CBD1 that results in a compaction of the CBDs and that probably reduces tension on the linker regions to the CLD. C, binding of Ca2+ to CBD2 allows sustained Na+/Ca2+ exchange and removes counteracting Na+-dependent inactivation. Whether Ca2+ binding to CBD2 induces yet another conformational change or just promotes an interaction with the CLD remains elusive. D, depiction of the PIP2-activated state of NCX that is independent of Ca2+ binding the CBDs.
      Recently, several reports have suggested that Ca2+ ions in sited I and II of CBD1 are not essential for Ca2+ regulation (
      • Chaptal V.
      • Ottolia M.
      • Mercado-Besserer G.
      • Nicoll D.A.
      • Philipson K.D.
      • Abramson J.
      Structure and functional analysis of a Ca2+ sensor mutant of the Na+/Ca2+ exchanger.
      ,
      • Ottolia M.
      • Nicoll D.A.
      • Philipson K.D.
      Roles of two Ca2+-binding domains in regulation of the cardiac Na+/Ca2+ exchanger.
      ). Although the impact of the Ca2+ ions in sites III and IV (Fig. 1F) is without doubt most dominant due to their proximity to CBD2, the lack of Ca2+-binding sites I and II would substantially change the electrostatic potential at the CBD1 Ca2+-binding sites. The consequence would be a reduction in the repulsion and attraction between the CBD1 Ca2+-binding sites and CBD2 in the absence and presence of Ca2+, respectively, yet these predicted effects are not reflected in functional studies with NCX1 mutants D421A and E454K (
      • Chaptal V.
      • Ottolia M.
      • Mercado-Besserer G.
      • Nicoll D.A.
      • Philipson K.D.
      • Abramson J.
      Structure and functional analysis of a Ca2+ sensor mutant of the Na+/Ca2+ exchanger.
      ,
      • Ottolia M.
      • Nicoll D.A.
      • Philipson K.D.
      Roles of two Ca2+-binding domains in regulation of the cardiac Na+/Ca2+ exchanger.
      ). Nevertheless, it remains intriguing why sites I and II should not be essential even though all Ca2+-coordinating residues in CBD1 are strictly conserved.
      To sustain Na+/Ca2+ exchange, Ca2+ ions probably have to also bind to CBD2 (Fig. 3C), a process that is isoform- and splice variant-specific. Functional (
      • Dunn J.
      • Elias C.L.
      • Le H.D.
      • Omelchenko A.
      • Hryshko L.V.
      • Lytton J.
      The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms.
      ,
      • Besserer G.M.
      • Ottolia M.
      • Nicoll D.A.
      • Chaptal V.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
      ,
      • Ottolia M.
      • Nicoll D.A.
      • Philipson K.D.
      Roles of two Ca2+-binding domains in regulation of the cardiac Na+/Ca2+ exchanger.
      ) and thermodynamic (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ) data for exchangers that bind zero to three Ca2+ ions at the CBD2 Ca2+-binding sites, as well as distinct macroscopic Ca2+ binding constants ranging from 250 nm to 20 μm, strongly indicate that the number and affinities of Ca2+-binding sites in CBD2 determine the form of the steady-state exchange current. For instance, exon B-encoded NCX1 splice variants become inactivated in a matter of seconds (
      • Dunn J.
      • Elias C.L.
      • Le H.D.
      • Omelchenko A.
      • Hryshko L.V.
      • Lytton J.
      The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms.
      ), whereas the NCX1 mutant K585E, which is comparable with the B variant of NCX3 and likely binds two Ca2+ ions between the CD and EF loops, shows steady-state outward currents above their corresponding peak currents (
      • Besserer G.M.
      • Ottolia M.
      • Nicoll D.A.
      • Chaptal V.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
      ,
      • Ottolia M.
      • Nicoll D.A.
      • Philipson K.D.
      Roles of two Ca2+-binding domains in regulation of the cardiac Na+/Ca2+ exchanger.
      ). At the same time, this contradicts suggestions that Ca2+ ions between the CD and EF loops of CBD2 are not essential either (
      • Besserer G.M.
      • Ottolia M.
      • Nicoll D.A.
      • Chaptal V.
      • Cascio D.
      • Philipson K.D.
      • Abramson J.
      The second Ca2+-binding domain of the Na+/Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.
      ), thus implying isoform- and splice variant-specific determinants to be irrelevant. In contrast, different properties of the CBD2 Ca2+-binding sites may enable optimal adaptation of Na+/Ca2+ exchange to meet special needs within the cell or a specific tissue.

      NCX Inactivation

      As cytosolic Na+ concentrations are low in normal functioning cells, it is unlikely that Na+-dependent inactivation plays a substantial role under normal physiological conditions. It could, however, be beneficial by limiting NCX-mediated Ca2+ influx under conditions such as ischemia, in which cytosolic Na+ levels are elevated and phosphatidylinositol 4,5-bisphosphate (PIP2) levels are low (
      • Reeves J.P.
      • Condrescu M.
      Ionic regulation of the cardiac sodium/calcium exchanger.
      ).
      As early as 1968, Baker and Blaustein (
      • Baker P.F.
      • Blaustein M.P.
      Sodium-dependent uptake of calcium by crab nerve.
      ) reported that a drop in cytosolic pH could inhibit Na+/Ca2+ exchange. Extensive studies on the effect of pH were later performed on sarcolemmal vesicles of canine heart (
      • Philipson K.D.
      • Bersohn M.M.
      • Nishimoto A.Y.
      Effects of pH on Na+/Ca2+ exchange in canine cardiac sarcolemmal vesicles.
      ) and guinea pig ventricular cells (
      • Hilgemann D.W.
      • Matsuoka S.
      • Nagel G.A.
      • Collins A.
      Steady-state and dynamic properties of cardiac sodium/calcium exchange. Sodium-dependent inactivation.
      ,
      • Doering A.E.
      • Lederer W.J.
      The mechanism by which cytoplasmic protons inhibit the sodium/calcium exchanger in guinea pig heart cells.
      ,
      • Doering A.E.
      • Eisner D.A.
      • Lederer W.J.
      Cardiac sodium/calcium exchange and pH.
      ). Prompted by the proton insensitivity of the transmembrane domain (
      • Doering A.E.
      • Lederer W.J.
      The mechanism by which cytoplasmic protons inhibit the sodium/calcium exchanger in guinea pig heart cells.
      ), experiments with the CBDs revealed that a mild reduction in cytosolic pH from 7.2 to 6.9 caused an up to 3-fold decrease in the Ca2+ affinities of CBD1 (
      • Boyman L.
      • Hagen B.M.
      • Giladi M.
      • Hiller R.
      • Lederer W.J.
      • Khananshvili D.
      Proton-sensing Ca2+-binding domains regulate the cardiac Na+/Ca2+ exchanger.
      ). This implies that pH is an additional regulator of NCX activity, as it efficiently modulates Ca2+ affinities of the CBD1 Ca2+-binding sites.
      In addition to its primary function as a Ca2+ efflux mechanism, NCX may also mediate Ca2+ influx, where high cytosolic Na+ concentrations induce a mode of exchange activity that does not require binding of Ca2+ to the CBDs (
      • Reeves J.P.
      • Condrescu M.
      Ionic regulation of the cardiac sodium/calcium exchanger.
      ,
      • Urbanczyk J.
      • Chernysh O.
      • Condrescu M.
      • Reeves J.P.
      Sodium/calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations.
      ). Under these conditions, counteracting Na+-dependent inactivation is overcome by high levels of PIP2, which keep NCX activated (Fig. 3D). Furthermore, it has been shown that PIP2 strongly interacts with residues 218–237 of NCX and that peptides encoding these residues can inhibit the exchanger (
      • Li Z.
      • Nicoll D.A.
      • Collins A.
      • Hilgemann D.W.
      • Filoteo A.G.
      • Penniston J.T.
      • Weiss J.N.
      • Tomich J.M.
      • Philipson K.D.
      Identification of a peptide inhibitor of the cardiac sarcolemmal Na+/Ca2+ exchanger.
      ). As a result, this region, which also shows some similarity to the calmodulin-binding motif, is referred to as the exchanger inhibitory peptide region. Mutational studies of the exchanger inhibitory peptide region in intact NCX revealed a profound effect of changes in sequence with respect to the interaction with PIP2 as well as Na+-dependent inactivation (
      • He Z.
      • Feng S.
      • Tong Q.
      • Hilgemann D.W.
      • Philipson K.D.
      Interaction of PIP2 with the XIP region of the cardiac sodium/calcium exchanger.
      ).

      NCX in Disease

      NCX is of paramount importance in the heart, as, together with the sarcoplasmic reticulum Ca2+-ATPase, the exchanger removes ∼99% (∼30% via NCX and ∼70% via sarcoplasmic reticulum Ca2+-ATPase) of the Ca2+ released to the cytosol (
      • Bers D.M.
      Cardiac excitation-contraction coupling.
      ). Dysregulation of this Ca2+ balance can lead to cardiac arrhythmias, a leading cause of death in cardiovascular disease. In the failing heart and in patients with ischemia-reperfusion injury (
      • Pignataro G.
      • Gala R.
      • Cuomo O.
      • Tortiglione A.
      • Giaccio L.
      • Castaldo P.
      • Sirabella R.
      • Matrone C.
      • Canitano A.
      • Amoroso S.
      • Di Renzo G.
      • Annunziato L.
      Two sodium/calcium exchanger gene products, NCX1 and NCX3, play a major role in the development of permanent focal cerebral ischemia.
      ,
      • Lee C.
      • Dhalla N.S.
      • Hryshko L.V.
      Therapeutic potential of novel Na+/Ca2+ exchange inhibitors in attenuating ischemia-reperfusion injury.
      ), in cases of salt-sensitive hypertension (
      • Iwamoto T.
      • Kita S.
      • Zhang J.
      • Blaustein M.P.
      • Arai Y.
      • Yoshida S.
      • Wakimoto K.
      • Komuro I.
      • Katsuragi T.
      Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type 1 in vascular smooth muscle.
      ,
      • Blaustein M.P.
      • Zhang J.
      • Chen L.
      • Hamilton B.P.
      How does salt retention raise blood pressure?.
      ) or multiple sclerosis (
      • Craner M.J.
      • Newcombe J.
      • Black J.A.
      • Hartle C.
      • Cuzner M.L.
      • Waxman S.G.
      Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na+/Ca2+ exchanger.
      ), NCX appears to operate predominantly in reverse mode, importing rather than exporting Ca2+. The resulting excessive Ca2+ entry may cause the activation of Ca2+-dependent proteases such as calpain and caspases (
      • Bano D.
      • Young K.W.
      • Guerin C.J.
      • Lefeuvre R.
      • Rothwell N.J.
      • Naldini L.
      • Rizzuto R.
      • Carafoli E.
      • Nicotera P.
      Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity.
      ), subsequently leading to cell apoptosis. As the damaging role of NCX is related mostly to its reverse mode, benzyloxyphenyl derivatives (
      • Iwamoto T.
      Forefront of Na+/Ca2+ exchanger studies: molecular pharmacology of Na+/Ca2+ exchange inhibitors.
      ) such as KB-R7943 (
      • Iwamoto T.
      • Watano T.
      • Shigekawa M.
      A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1.
      ,
      • Iwamoto T.
      • Kita S.
      Development and application of Na+/Ca2+ exchange inhibitors.
      ), SEA0400 (
      • Iwamoto T.
      • Kita S.
      • Uehara A.
      • Imanaga I.
      • Matsuda T.
      • Baba A.
      • Katsuragi T.
      Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400.
      ), and SN-6 (
      • Iwamoto T.
      • Inoue Y.
      • Ito K.
      • Sakaue T.
      • Kita S.
      • Katsuragi T.
      The exchanger inhibitory peptide region-dependent inhibition of Na+/Ca2+ exchange by SN-6 (2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester), a novel benzyloxyphenyl derivative.
      ), which specifically inhibit the reverse mode operation of NCX, have attracted a great deal of attention (for review, see Ref.
      • Annunziato L.
      • Pignataro G.
      • Di Renzo G.F.
      Pharmacology of brain Na+/Ca2+ exchanger: from molecular biology to therapeutic perspectives.
      ). Although experiences with these inhibitors are overall rather positive, thorough testing in relevant preclinical models remains to be performed (
      • Antoons G.
      • Willems R.
      • Sipido K.R.
      Alternative strategies in arrhythmia therapy: evaluation of sodium/calcium exchange as an anti-arrhythmic target.
      ).

      Concluding Remarks

      Over the last 5 years, high resolution NMR and x-ray structures of a large part of the regulatory loop of NCX and the transmembrane domain of a prokaryotic NCX homolog have added a new dimension to our understanding of the regulation and function of this essential transporter. In addition, the role of different splice variants has become increasingly clear. The variability in CBD2 as a consequence of alternative splicing is now known to modulate not only the number and affinities of the Ca2+-binding sites in CBD2 but also the Ca2+ affinities in CBD1 (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ). Furthermore, the CBD12 SAXS solution structures have suggested a conformational change in the hinge between CBD1 and CBD2 upon Ca2+ binding (
      • Hilge M.
      • Aelen J.
      • Foarce A.
      • Perrakis A.
      • Vuister G.W.
      Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
      ), whereas CBD12 x-ray structures have revealed the link between the CBD1 Ca2+-binding sites and CBD2 (
      • Wu M.
      • Tong S.
      • Gonzalez J.
      • Jayaraman V.
      • Spudich J.L.
      • Zheng L.
      Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing.
      ). This description of the interface includes hydrogen bonds and salt bridges with residues located in the FG loop of CBD2. Future efforts will aim to connect the regulatory effects of the CBDs to the ion-conducting transmembrane domain of NCX.

      Acknowledgments

      I thank Dr. Shirley A. Müller for critically reading the manuscript and Stephan Hilge for professional help with the figures.

      Supplementary Material

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