title: Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation.
abstract: Na(+)/Ca(2+) exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca(2+) homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na(+) or Ca(2+), and thus can catalyze uphill Ca(2+) transport driven by a Na(+) gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na(+) ions occupy the so-called Sext, SCa, and Sint sites, whereas the Smid site is occupied by one water molecule and one H(+), which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na(+)/Ca(2+) and Ca(2+)/Ca(2+) exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na(+) and Ca(2+) requires protonation of D240, and that this side chain does not coordinate either ion at Smid. These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na(+) across the membrane is electrogenic, whereas transport of Ca(2+) is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.
authors.affiliation: Theoretical Molecular Biophysics Section, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Marinelli
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Lior
authors.initials: L
authors.lastname: Almagor
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Reuben
authors.initials: R
authors.lastname: Hiller
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Moshe
authors.initials: M
authors.lastname: Giladi
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel jose.faraldo@nih.gov dhanan@post.tau.ac.il.
authors.forename: Daniel
authors.initials: D
authors.lastname: Khananshvili
authors.affiliation: Theoretical Molecular Biophysics Section, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and jose.faraldo@nih.gov dhanan@post.tau.ac.il.
authors.forename: José D
authors.initials: JD
authors.lastname: Faraldo-Gómez
mesh_terms: D004926:Escherichia coli; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D063747:Methanocaldococcus / Q000235:genetics*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D019831:Sodium-Calcium Exchanger; D012964:Sodium
keywords: CaCA superfamily; ion specificity; membrane antiporters; molecular-dynamics simulations; secondary transporters