Structure-Dynamic Coupling Through Ca2+-Binding Regulatory Domains of Mammalian NCX Isoform/Splice Variants

  • Daniel KhananshviliEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 981)


Mammalian Na+/Ca2+ exchangers (NCX1, NCX2, and NCX3) and their splice variants are expressed in a tissue-specific manner and are regulated by Ca2+ binding CBD1 and CBD2 domains. NCX2 does not undergo splicing, whereas in NCX1 and NCX3, the splicing segment (with mutually exclusive and cassette exons) is located in CBD2. Ca2+ binding to CBD1 results in Ca2+-dependent tethering of CBDs through the network of interdomain salt-bridges, which is associated with NCX activation, whereas a slow dissociation of “occluded” Ca2+ inactivates NCX. Although NCX variants share a common structural basis for Ca2+-dependent tethering of CBDs, the Ca2+ off-rates of occluded Ca2+ vary up to 50-fold, depending on the exons assembly. The Ca2+-dependent tethering of CBDs rigidifies the interdomain movements of CBDs without any significant changes in the CBDs’ alignment; consequently, more constraining conformational states become more populated in the absence of global conformational changes. Although this Ca2+-dependent “population shift” is a common mechanism among NCX variants, the strength and span of backbone rigidification from the C-terminal of CBD1 to the C-terminal of CBD2 is exon dependent. The mutually exclusive exons differentially stabilize/destabilize the backbone dynamics of Ca2+-bound CBDs in NCX1 and NCX3 variants, whereas the cassette exons control the stability of the interdomain linker. The combined effects of mutually exclusive and cassette exons permit a fine adjustment of two different regulatory pathways: the Ca2+-dependent activation (controlled by CBD1) and the Ca2+-dependent alleviation of Na+-induced inactivation (controlled by CBD2). Exon-controlled dynamic features match with cell-specific regulatory requirements in a given variant.


NCX SAXS HDX-MS Dynamic coupling Population shift Allosteric regulation Alternative splicing Exon 



This work was supported by the Israel Science Foundation Grant #825/14 to DK. The financial support of the Fields Estate foundation to DK is highly appreciated.


  1. 1.
    Carafoli E, Krebs J (2016) Why calcium? How calcium became the best communicator. J Biol Chem 291:20849–20857CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Krebs J (2009) The influence of calcium signaling on the regulation of alternative splicing. Biochim Biophys Acta 1793:979–984CrossRefPubMedGoogle Scholar
  3. 3.
    McCue HV, Haynes LP, Burgoyne RD (2010) The diversity of calcium sensor proteins in the regulation of neuronal function. Cold Spring Harb Perspect Biol 2(8):a004085CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Khananshvili D (2013) The SLC8 gene family of sodium-calcium exchangers (NCX) – structure, function, and regulation in health and disease. Mol Asp Med 34:220–235CrossRefGoogle Scholar
  5. 5.
    Khananshvili D (2014) Sodium-calcium exchangers (NCX): molecular hallmarks underlying the tissue-specific and systemic functions. Pflugers Arch 466:43–60CrossRefPubMedGoogle Scholar
  6. 6.
    Brini M, Calì T, Ottolini D, Carafoli E (2014) The plasma membrane calcium pump in health and disease. FEBS J 280:5385–5397CrossRefGoogle Scholar
  7. 7.
    Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs. Biochem J 405:199–221CrossRefPubMedGoogle Scholar
  8. 8.
    Plattner H, Verkhratsky A (2016) Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling. Philos Trans R Soc Lond Ser B Biol Sci 371:1700CrossRefGoogle Scholar
  9. 9.
    Williams RJP (1999) Calcium: the developing role of its chemistry in biological evolution. In: Carafoli E, Klee C (eds) Calcium as a cellular regulator. Oxford University Press, New York, pp 3–27Google Scholar
  10. 10.
    Nussinov R, Tsai CJ (2013) Allostery in disease and in drug discovery. Cell 153:293–305CrossRefPubMedGoogle Scholar
  11. 11.
    Rader AJ, Brown SM (2011) Correlating allostery with rigidity. Mol BioSyst 7:464–471CrossRefPubMedGoogle Scholar
  12. 12.
    Tsai CJ, Nussinov R (2014) A unified view of “how allostery works”. PLoS Comput Biol 10:e1003394CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Khananshvili D (2016) Regulation of Ca2+-ATPases, V-ATPases and F-ATPases. In: Chakraborti S, Dhalla NS (eds) Advances in biochemistry in health and disease, vol 14. Springer, Cham, pp 93–116Google Scholar
  14. 14.
    Philipson KD, Nicoll DA (2000) Sodium-calcium exchange: a molecular perspective. Annu Rev Physiol 62:111–133CrossRefPubMedGoogle Scholar
  15. 15.
    Ren X, Philipson KD (2013) The topology of the cardiac Na+/Ca2+ exchanger, NCX1. J Mol Cell Cardiol 57:68–71CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Lytton J (2007) Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem J 406:365–382CrossRefPubMedGoogle Scholar
  17. 17.
    Nicoll DA, Longoni S, Philipson KD (1990) Molecular cloning and functional expression of the cardiac sarcolemmal Na+-Ca2+ exchanger. Science 250:562–565CrossRefPubMedGoogle Scholar
  18. 18.
    Quednau BD, Nicoll DA, Philipson KD (1997) Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat. Am J Phys 272:C1250–C1261CrossRefGoogle Scholar
  19. 19.
    Liao J, Li H, Zeng W, Sauer DB, Belmares R, Jiang Y (2012) Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger. Science 335:686–690CrossRefPubMedGoogle Scholar
  20. 20.
    Liao J, Marinelli F, Lee C, Huang Y, Faraldo-Gómez JD, Jiang Y (2016) Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger. Nat Struct Mol Biol 23:590–599CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Almagor L, Giladi M, van Dijk L, Buki T, Hiller R, Khananshvili D (2014) Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium-calcium exchanger (NCX_Mj). Cell Calcium 56:276–284CrossRefPubMedGoogle Scholar
  22. 22.
    Giladi M, Almagor L, van Dijk L, Hiller R, Man P, Forest E, Khananshvili D (2016) Asymmetric preorganization of inverted pair residues in the sodium–calcium exchanger. Sci Rep 6:20753CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Giladi M, Tal I, Khananshvili D (2016) Structural features of ion transport and allosteric regulation in sodium-calcium exchanger (NCX) proteins. Front Physiol 7(30):1–13Google Scholar
  24. 24.
    Marinelli F, Almagor L, Hiller R, Giladi M, Khananshvili D, Faraldo-Gomez JD (2014) Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation. Proc Natl Acad Sci USA 111:E5354–E5362CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hilge M, Aelen J, Foarce A, Perrakis A, Vuister GW (2009) Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism. Proc Natl Acad Sci USA 106:14333–14338CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hilge M, Aelen J, Vuister GW (2006) Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors. Mol Cell 22:15–25CrossRefPubMedGoogle Scholar
  27. 27.
    Palty R, Silverman WF, Hershfinkel M, Caporale T, Sensi SL, Parnis J, Nolte C, Fishman D, Shoshan-Barmatz V, Herrmann S, Khananshvili D, Sekler I (2010) NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. Proc Natl Acad Sci USA 107:436–441CrossRefPubMedGoogle Scholar
  28. 28.
    Besserer GM, Ottolia M, Nicoll DA, Chaptal V, Cascio D, Philipson KD, Abramson J (2007) The second Ca2+-binding domain of the Na+ Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis. Proc Natl Acad Sci USA 104:18467–18472CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Giladi M, Sasson Y, Fang X, Hiller R, Buki T, Wang YX, Hirsch JA, Khananshvili D (2012c) A common Ca2+-driven interdomain module governs eukaryotic NCX regulation. PLoS One 7:e39985CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Nicoll DA, Sawaya MR, Kwon S, Cascio D, Philipson KD, Abramson J (2006) The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif. J Biol Chem 281:21577–21581CrossRefPubMedGoogle Scholar
  31. 31.
    Wu M, Le HD, Wang M, Yurkov V, Omelchenko A, Hnatowich M, Nix J, Hryshko LV, Zheng L (2010) 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. J Biol Chem 285:2554–2561CrossRefPubMedGoogle Scholar
  32. 32.
    Wu M, Tong S, Gonzalez J, Jayaraman V, Spudich JL, Zheng L (2011) Structural basis of the Ca2+ inhibitory mechanism of Drosophila Na+/Ca2+ exchanger CALX and its modification by alternative splicing. Structure 19:1509–1517CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Tal I, Kozlovsky T, Brisker D, Giladi M, Khananshvili D (2016) Kinetic and equilibrium properties of regulatory Ca2+-binding domains in sodium-calcium exchangers 2 and 3. Cell Calcium 59:181–188CrossRefPubMedGoogle Scholar
  34. 34.
    Giladi M, Bohbot H, Buki T, Schulze DH, Hiller R, Khananshvili D (2012) Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants. Cell Calcium 51:478–485CrossRefPubMedGoogle Scholar
  35. 35.
    Giladi M, Boyman L, Mikhasenko H, Hiller R, Khananshvili D (2010) Essential role of the CBD1-CBD2 linker in slow dissociation of Ca2+ from the regulatory two-domain tandem of NCX1. J Biol Chem 285:28117–28125CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Giladi M, Friedberg I, Fang X, Hiller R, Wang YX, Khananshvili D (2012) G503 is obligatory for coupling of regulatory domains in NCX proteins. Biochemistry 51:7313–7320CrossRefPubMedGoogle Scholar
  37. 37.
    Dyck C, Maxwell K, Buchko J, Trac M, Omelchenko A, Hnatowich M, Hryshko LV (1998) Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites. J Biol Chem 273:12981–12987CrossRefPubMedGoogle Scholar
  38. 38.
    Dyck C, Omelchenko A, Elias CL, Quednau BD, Philipson KD, Hnatowich M, Hryshko LV (1999) Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na+-Ca2+ exchanger. J Gen Physiol 114:701–711CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Hilgemann DW, Matsuoka S, Nagel GA, Collins A (1992) Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation. J Gen Physiol 100:905–932CrossRefPubMedGoogle Scholar
  40. 40.
    Boyman L, Mikhasenko H, Hiller R, Khananshvili D (2009) Kinetic and equilibrium properties of regulatory calcium sensors of NCX1 protein. J Biol Chem 284:6185–6193CrossRefPubMedGoogle Scholar
  41. 41.
    Boyman L, Hagen BM, Giladi M, Hiller R, Lederer WJ, Khananshvili D (2011) Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger. J Biol Chem 286:28811–28820CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Hryshko LV, Matsuoka S, Nicoll DA, Weiss JN, Schwarz EM, Benzer S, Philipson KD (1996) Anomalous regulation of the Drosophila Na+-Ca2+ exchanger by Ca2+. J Gen Physiol 108:67–74CrossRefPubMedGoogle Scholar
  43. 43.
    Matsuoka S, Nicoll DA, Hryshko LV, Levitsky DO, Weiss JN, Philipson KD (1995) Regulation of the cardiac Na+-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+ binding domain. J Gen Physiol 105:403–420CrossRefPubMedGoogle Scholar
  44. 44.
    Ottolia M, Nicoll DA, Philipson KD (2009) Roles of two Ca2+-binding domains in regulation of the cardiac Na+-Ca2+ exchanger. J Biol Chem 284:32735–32741CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Breukels V, Konijnenberg A, Nabuurs SM, Touw WG, Vuister GW (2011) The second Ca2+-binding domain of NCX1 binds Mg2+ with high affinity. Biochemistry 50:8804–8812CrossRefPubMedGoogle Scholar
  46. 46.
    DiPolo R, Beauge L (2006) Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions. Physiol Rev 86:155–203CrossRefPubMedGoogle Scholar
  47. 47.
    Omelchenko A, Dyck C, Hnatowich M, Buchko J, Nicoll DA, Philipson KD, Hryshko LV (1998) Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. J Gen Physiol 111:691–702CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    John SA, Ribalet B, Weiss JN, Philipson KD, Ottolia M (2011) Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers. Proc Natl Acad Sci USA 108:1699–1704CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Giladi M, Hiller R, Hirsch JA, Khananshvili D (2013) Population shift underlies Ca2+-induced regulatory transitions in the sodium-calcium exchanger (NCX). J Biol Chem 288:23141–23149CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Giladi M, Lee SY, Ariely Y, Teldan Y, Granit R, Strulevich R, Haitin Y, Chung KY, Khananshvili D (2017) Structure-based dynamic arrays in regulatory domains of sodium-calcium exchanger (NCX) isoforms. Sci Rep 7(1):993CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Giladi M, Lee SY, Hiller R, Chung KY, Khananshvili D (2015) Structure-dynamic determinants governing a mode of regulatory response and propagation of allosteric signal in splice variants of Na+/Ca2+ exchange (NCX) proteins. Biochem J 465:489–501CrossRefPubMedGoogle Scholar
  52. 52.
    Lee SY, Giladi M, Bohbot H, Hiller R, Chung KY, Khananshvili D (2016) Structure-dynamic basis of splicing dependent regulation in tissue-specific variants of the sodium-calcium exchanger (NCX1). FASEB J 30:1356–1366CrossRefPubMedGoogle Scholar
  53. 53.
    Abiko LA, Vitale PM, Favaro DC, Hauk P, Li DW, Yuan J, Bruschweiler-Li L, Salinas RK, Brüschweiler R (2016) Model for the allosteric regulation of the Na+/Ca2+ exchanger (NCX). Proteins 84:580–590CrossRefPubMedGoogle Scholar
  54. 54.
    Salinas RK, Bruschweiler-Li L, Johnson E, Bruschweiler R (2011) Ca2+ binding alters the interdomain flexibility between the two cytoplasmic calcium-binding domains in the Na+/Ca2+ exchanger. J Biol Chem 286:32123–32131CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Buljan M, Chalancon G, Dunker AK, Bateman A, Balaji S, Fuxreiter M, Babu MM (2013) Alternative splicing of intrinsically disordered regions and rewiring of protein interactions. Curr Opin Struct Biol 23:443–450CrossRefPubMedGoogle Scholar
  56. 56.
    Latysheva NS, Flock T, Weatheritt RJ, Chavali S, Babu MM (2015) How do disordered regions achieve comparable functions to structured domains? Protein Sci 24:909–922CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Physiology and Pharmacology, Sackler School of MedicineTel-Aviv UniversityTel-AvivIsrael

Personalised recommendations