Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

Blavatnik Center for Drug Discovery Tel Aviv University Tel Aviv 69978 Israel

Department of Biochemistry Faculty of Science Charles University 128 00 Prague Czech Republic

Department of Physiology and Pharmacology Faculty of Medicine Tel Aviv University Tel Aviv 69978 Israel

Division BioCeV Institute of Microbiology of the Czech Academy of Sciences Prumyslova 595 252 50 Vestec Prague Czech Republic

Tel Aviv Sourasky Medical Center Tel Aviv 6423906 Israel

See more in PubMed

Philipson KD, Nicoll DA. Sodium-calcium exchange: a molecular perspective. Annu. Rev. Physiol. 2000;62:111–133. doi: 10.1146/annurev.physiol.62.1.111. PubMed DOI

Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol. Rev. 1999;79:763–854. doi: 10.1152/physrev.1999.79.3.763. PubMed DOI

Lytton J. Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem. J. 2007;406:365–382. doi: 10.1042/BJ20070619. PubMed DOI

Khananshvili D. Basic and editing mechanisms underlying ion transport and regulation in NCX variants. Cell Calcium. 2020;85:102131. doi: 10.1016/j.ceca.2019.102131. PubMed DOI

Hilgemann DW. Regulation of ion transport from within ion transit pathways. J. Gen. Physiol. 2020;152:e201912455. doi: 10.1085/jgp.201912455. PubMed DOI PMC

DiPolo R, Beaugé L. Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions. Physiol. Rev. 2006;86:155–203. doi: 10.1152/physrev.00018.2005. PubMed DOI

Ottolia M, John S, Hazan A, Goldhaber JI. The cardiac Na+-Ca2+ exchanger: from structure to function. Compr. Physiol. 2021;12:2681–2717. doi: 10.1002/cphy.c200031. PubMed DOI PMC

Giladi M, Tal I, Khananshvili D. Structural features of ion transport and allosteric regulation in Sodium-Calcium Exchanger (NCX) proteins. Front. Physiol. 2016;7:30. doi: 10.3389/fphys.2016.00030. PubMed DOI PMC

Khananshvili D. Structure-based function and regulation of NCX variants: updates and challenges. Int. J. Mol. Sci. 2022;24:61. doi: 10.3390/ijms24010061. PubMed DOI PMC

Liao J, et al. Structural insight into the ion-exchange mechanism of the sodium/calcium exchanger. Science. 2012;335:686–690. doi: 10.1126/science.1215759. PubMed DOI

Liao J, et al. Mechanism of extracellular ion exchange and binding-site occlusion in a sodium/calcium exchanger. Nat. Struct. Mol. Biol. 2016;23:590–599. doi: 10.1038/nsmb.3230. PubMed DOI PMC

Almagor L, et al. Functional asymmetry of bidirectional Ca2+-movements in an archaeal sodium-calcium exchanger (NCX_Mj) Cell Calcium. 2014;56:276–284. doi: 10.1016/j.ceca.2014.08.010. PubMed DOI

Marinelli F, et al. Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation. Proc. Natl Acad. Sci. USA. 2014;111:E5354–E5362. doi: 10.1073/pnas.1415751111. PubMed DOI PMC

Giladi M, et al. Asymmetric preorganization of inverted pair residues in the sodium-calcium exchanger. Sci. Rep. 2016;6:20753. doi: 10.1038/srep20753. PubMed DOI PMC

Giladi M, et al. Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states. J. Biol. Chem. 2017;292:12311–12323. doi: 10.1074/jbc.M117.787168. PubMed DOI PMC

van Dijk L, et al. Key residues controlling bidirectional ion movements in Na+/Ca2+ exchanger. Cell Calcium. 2018;76:10–22. doi: 10.1016/j.ceca.2018.09.004. PubMed DOI PMC

Khananshvili D. The Archaeal Na+/Ca2+ Exchanger (NCX_Mj) as a model of ion transport for the superfamily of Ca2+/CA antiporters. Front. Chem. 2021;9:722336. doi: 10.3389/fchem.2021.722336. PubMed DOI PMC

Li Z, et al. Identification of a peptide inhibitor of the cardiac sarcolemmal Na+-Ca2+ exchanger. J. Biol. Chem. 1991;266:1014–1020. doi: 10.1016/S0021-9258(17)35276-6. PubMed DOI

Matsuoka S, Nicoll DA, He Z, Philipson KD. Regulation of cardiac Na+-Ca2+ exchanger by the endogenous XIP region. J. Gen. Physiol. 1997;109:273–286. doi: 10.1085/jgp.109.2.273. PubMed DOI PMC

Yuan, J., Yuan, C., Xie, M., Yu, L., Bruschweiler, L. & Brüschweiler, R. The intracellular loop of the Na+/Ca2+ exchanger contains an ‘awareness ribbon’-shaped two-helix bundle domain. Biochemistry. 57, 5096–5104, PubMed. https://pubmed.ncbi.nlm.nih.gov/29898361/ (2018). PubMed

Hilge M, Aelen J, Vuister GW. Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors. Mol. Cell. 2006;22:15–25. doi: 10.1016/j.molcel.2006.03.008. PubMed DOI

Hilge M, Aelen J, Foarce A, Perrakis A, Vuister GW. Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism. Proc. Natl Acad. Sci. USA. 2009;106:14333–14338. doi: 10.1073/pnas.0902171106. PubMed DOI PMC

Nicoll DA, et al. The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif. J. Biol. Chem. 2006;281:21577–21581. doi: 10.1074/jbc.C600117200. PubMed DOI

Besserer GM, et al. The second Ca2+-binding domain of the Na+-Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis. Proc. Natl Acad. Sci. USA. 2007;104:18467–18472. doi: 10.1073/pnas.0707417104. PubMed DOI PMC

Gök C, Fuller W. Regulation of NCX1 by palmitoylation. Cell Calcium. 2020;86:102158. doi: 10.1016/j.ceca.2019.102158. PubMed DOI

Gök C, et al. Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1. Cell Calcium. 2021;97:102408. doi: 10.1016/j.ceca.2021.102408. PubMed DOI PMC

Main A, Fuller W. Protein S-Palmitoylation: advances and challenges in studying a therapeutically important lipid modification. FEBS J. 2022;289:861–882. doi: 10.1111/febs.15781. PubMed DOI

Hilgemann DW, Matsuoka S, Nagel GA, Collins A. Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation. J. Gen. Physiol. 1992;100:905–932. doi: 10.1085/jgp.100.6.905. PubMed DOI PMC

Hilgemann DW. Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches. Nature. 1990;344:242–245. doi: 10.1038/344242a0. PubMed DOI

Hryshko L. What regulates Na+/Ca2+ exchange? Focus on ‘Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells’. Am. J. Physiol. Cell Physiol. 2008;295:C869–C871. doi: 10.1152/ajpcell.00420.2008. PubMed DOI

He Z, Feng S, Tong Q, Hilgemann DW, Philipson KD. Interaction of PIP2 with the XIP region of the cardiac Na/Ca exchanger. Am. J. Physiol. Cell Physiol. 2000;278:C661–C666. doi: 10.1152/ajpcell.2000.278.4.C661. PubMed DOI

Shen C, et al. Dual control of cardiac Na+-Ca2+ exchange by PIP2: analysis of the surface membrane fraction by extracellular cysteine PEGylation. J. Physiol. 2007;582:1011–1026. doi: 10.1113/jphysiol.2007.132720. PubMed DOI PMC

Boyman L, et al. Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger. J. Biol. Chem. 2011;286:28811–28820. doi: 10.1074/jbc.M110.214106. PubMed DOI PMC

Linck B, et al. Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3) Am. J. Physiol. 1998;274:C415–C423. doi: 10.1152/ajpcell.1998.274.2.C415. PubMed DOI

Giladi M, et al. A common Ca2+-driven interdomain module governs eukaryotic NCX regulation. PLoS One. 2012;7:e39985. doi: 10.1371/journal.pone.0039985. PubMed DOI PMC

Giladi M, Boyman L, Mikhasenko H, Hiller R, Khananshvili D. Essential role of the CBD1-CBD2 linker in slow dissociation of Ca2+ from the regulatory two-domain tandem of NCX1. J. Biol. Chem. 2010;285:28117–28125. doi: 10.1074/jbc.M110.127001. PubMed DOI PMC

Giladi M, et al. Dynamic features of allosteric Ca2+ sensor in tissue-specific NCX variants. Cell Calcium. 2012;51:478–485. doi: 10.1016/j.ceca.2012.04.007. PubMed DOI

Boyman L, Mikhasenko H, Hiller R, Khananshvili D. Kinetic and equilibrium properties of regulatory calcium sensors of NCX1 protein. J. Biol. Chem. 2009;284:6185–6193. doi: 10.1074/jbc.M809012200. PubMed DOI

Giladi M, et al. Structure-based dynamic arrays in regulatory domains of sodium-calcium exchanger (NCX) isoforms. Sci. Rep. 2017;7:993. doi: 10.1038/s41598-017-01102-x. PubMed DOI PMC

Tal I, Kozlovsky T, Brisker D, Giladi M, Khananshvili D. Kinetic and equilibrium properties of regulatory Ca2+-binding domains in sodium-calcium exchangers 2 and 3. Cell Calcium. 2016;59:181–188. doi: 10.1016/j.ceca.2016.01.008. PubMed DOI

Abiko LA, et al. Model for the allosteric regulation of the Na+/Ca2+ exchanger NCX. Proteins. 2016;84:580–590. doi: 10.1002/prot.25003. PubMed DOI

Salinas RK, Bruschweiler-Li L, Johnson E, Brüschweiler R. Ca2+ binding alters the interdomain flexibility between the two cytoplasmic calcium-binding domains in the Na+/Ca2+ exchanger. J. Biol. Chem. 2011;286:32123–32131. doi: 10.1074/jbc.M111.249268. PubMed DOI PMC

Giladi M, Lee SY, Hiller R, Chung KY, Khananshvili D. 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. 2015;465:489–501. doi: 10.1042/BJ20141036. PubMed DOI

Lee SY, et al. Structure-dynamic basis of splicing-dependent regulation in tissue-specific variants of the sodium-calcium exchanger. FASEB J. 2016;30:1356–1366. doi: 10.1096/fj.15-282251. PubMed DOI

Giladi M, Hiller R, Hirsch JA, Khananshvili D. Population shift underlies Ca2+-induced regulatory transitions in the sodium-calcium exchanger (NCX) J. Biol. Chem. 2013;288:23141–23149. doi: 10.1074/jbc.M113.471698. PubMed DOI PMC

Dunn J, et al. The molecular determinants of ionic regulatory differences between brain and kidney Na+/Ca2+ exchanger (NCX1) isoforms. J. Biol. Chem. 2002;277:33957–33962. doi: 10.1074/jbc.M206677200. PubMed DOI

Dyck C, et al. Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na+-Ca2+ exchanger. J. Gen. Physiol. 1999;114:701–711. doi: 10.1085/jgp.114.5.701. PubMed DOI PMC

Michel LYM, et al. Function and regulation of the Na+-Ca2+ exchanger NCX3 splice variants in brain and skeletal muscle. J. Biol. Chem. 2014;289:11293–11303. doi: 10.1074/jbc.M113.529388. PubMed DOI PMC

Xue J, et al. Structural mechanisms of the human cardiac sodium-calcium exchanger NCX1. Nat. Commun. 2023;14:6181. doi: 10.1038/s41467-023-41885-4. PubMed DOI PMC

Dong Y, et al. Structural insight into the allosteric inhibition of human sodium-calcium exchanger NCX1 by XIP and SEA0400. EMBO J. 2024;43:14–31. doi: 10.1038/s44318-023-00013-0. PubMed DOI PMC

Giladi M, Khananshvili D. Hydrogen-Deuterium exchange mass-spectrometry of secondary active transporters: from structural dynamics to molecular mechanisms. Front. Pharm. 2020;11:70. doi: 10.3389/fphar.2020.00070. PubMed DOI PMC

Coppieters ’t Wallant K, Martens C. Hydrogen-deuterium exchange coupled to mass spectrometry: a multifaceted tool to decipher the molecular mechanism of transporters. Biochimie. 2023;205:95–101. doi: 10.1016/j.biochi.2022.08.014. PubMed DOI

Glasgow A, et al. Ligand-specific changes in conformational flexibility mediate long-range allostery in the lac repressor. Nat. Commun. 2023;14:1179. doi: 10.1038/s41467-023-36798-1. PubMed DOI PMC

Gourinchas G, et al. Long-range allosteric signaling in red light-regulated diguanylyl cyclases. Sci. Adv. 2017;3:e1602498. doi: 10.1126/sciadv.1602498. PubMed DOI PMC

Jia R, Bradshaw RT, Calvaresi V, Politis A. Integrating Hydrogen Deuterium exchange-mass spectrometry with molecular simulations enables quantification of the conformational populations of the sugar transporter XylE. J. Am. Chem. Soc. 2023;145:7768–7779. doi: 10.1021/jacs.2c06148. PubMed DOI PMC

Khananshvili D, Weil-Maslansky E, Baazov D. Kinetics and mechanism: modulation of ion transport in the cardiac sarcolemma sodium-calcium exchanger by protons, monovalent, ions, and temperature. Ann. N. Y Acad. Sci. 1996;779:217–235. doi: 10.1111/j.1749-6632.1996.tb44789.x. PubMed DOI

Hilgemann DW. Unitary cardiac Na+, Ca2+ exchange current magnitudes determined from channel-like noise and charge movements of ion transport. Biophys. J. 1996;71:759–768. doi: 10.1016/S0006-3495(96)79275-5. PubMed DOI PMC

Niggli E, Lederer WJ. Molecular operations of the sodium-calcium exchanger revealed by conformation currents. Nature. 1991;349:621–624. doi: 10.1038/349621a0. PubMed DOI

Baazov D, Wang X, Khananshvili D. Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe. Biochemistry. 1999;38:1435–1445. doi: 10.1021/bi981429u. PubMed DOI

Smith LJ, Daura X, van Gunsteren WF. Assessing equilibration and convergence in biomolecular simulations. Proteins: Struct. Funct. Bioinforma. 2002;48:487–496. doi: 10.1002/prot.10144. PubMed DOI

Nussinov R, Zhang M, Liu Y, Jang H. AlphaFold, allosteric, and orthosteric drug discovery: ways forward. Drug Discov. Today. 2023;28:103551. doi: 10.1016/j.drudis.2023.103551. PubMed DOI PMC

Nussinov R, Tsai C-J, Jang H. Allostery, and how to define and measure signal transduction. Biophys. Chem. 2022;283:106766. doi: 10.1016/j.bpc.2022.106766. PubMed DOI PMC

Wodak SJ, et al. Allostery in its many disguises: from theory to applications. Structure. 2019;27:566–578. doi: 10.1016/j.str.2019.01.003. PubMed DOI PMC

Giladi M, et al. G503 is obligatory for coupling of regulatory domains in NCX proteins. Biochemistry. 2012;51:7313–7320. doi: 10.1021/bi300739z. PubMed DOI

Ottolia M, Nicoll DA, Philipson KD. Mutational analysis of the alpha-1 repeat of the cardiac Na+-Ca2+ exchanger. J. Biol. Chem. 2005;280:1061–1069. doi: 10.1074/jbc.M411899200. PubMed DOI

Scranton K, John S, Escobar A, Goldhaber JI, Ottolia M. Modulation of the cardiac Na+-Ca2+ exchanger by cytoplasmic protons: molecular mechanisms and physiological implications. Cell Calcium. 2020;87:102140. doi: 10.1016/j.ceca.2019.102140. PubMed DOI PMC

John S, Kim B, Olcese R, Goldhaber JI, Ottolia M. Molecular determinants of pH regulation in the cardiac Na+-Ca2+ exchanger. J. Gen. Physiol. 2018;150:245–257. doi: 10.1085/jgp.201611693. PubMed DOI PMC

Doering AE, et al. Topology of a functionally important region of the cardiac Na+/Ca2+ exchanger. J. Biol. Chem. 1998;273:778–783. doi: 10.1074/jbc.273.2.778. PubMed DOI

Reilly L, et al. Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling. FASEB J. 2015;29:4532–4543. doi: 10.1096/fj.15-276493. PubMed DOI PMC

Covington, A. K., Paabo, M., Robinson, R. A. & Bates, R. G. Use of the glass electrode in Deuterium Oxide and the relation between the standardized Pd (Pa/Sub D/) scale and the operational ph in heavy water. Anal. Chem. 40: 700–706. 10.1021/ac60260a013 (1968).

Yang M, et al. Recombinant Nepenthesin II for Hydrogen/Deuterium Exchange Mass Spectrometry. Anal. Chem. 2015;87:6681–6687. doi: 10.1021/acs.analchem.5b00831. PubMed DOI

Trcka F, et al. Human stress-inducible Hsp70 has a high propensity to form ATP-dependent antiparallel dimers that are differentially regulated by cochaperone binding. Mol. Cell Proteom. 2019;18:320–337. doi: 10.1074/mcp.RA118.001044. PubMed DOI PMC

Kavan D, Man P. MSTools—Web based application for visualization and presentation of HXMS data. Int. J. Mass Spectrom. 2011;302:53–58. doi: 10.1016/j.ijms.2010.07.030. DOI

Perez-Riverol Y, et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50:D543–D552. doi: 10.1093/nar/gkab1038. PubMed DOI PMC

Jumper J, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Madhavi Sastry G, Adzhigirey M, Day T, Annabhimoju R, Sherman W. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J. Comput. Aided Mol. Des. 2013;27:221–234. doi: 10.1007/s10822-013-9644-8. PubMed DOI

Sievers F, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011;7:539. doi: 10.1038/msb.2011.75. PubMed DOI PMC

Lomize AL, Todd SC, Pogozheva ID. Spatial arrangement of proteins in planar and curved membranes by PPM 3.0. Protein Sci. 2022;31:209–220. doi: 10.1002/pro.4219. PubMed DOI PMC

Harrach MF, Drossel B. Structure and dynamics of TIP3P, TIP4P, and TIP5P water near smooth and atomistic walls of different hydroaffinity. J. Chem. Phys. 2014;140:174501. doi: 10.1063/1.4872239. PubMed DOI

Roos K, et al. OPLS3e: extending force field coverage for drug-like small molecules. J. Chem. Theory Comput. 2019;15:1863–1874. doi: 10.1021/acs.jctc.8b01026. PubMed DOI