Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation

Proc Natl Acad Sci U S A. 2014 Dec 16;111(50):E5354-62. doi: 10.1073/pnas.1415751111. Epub 2014 Dec 2.


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.

Keywords: CaCA superfamily; ion specificity; membrane antiporters; molecular-dynamics simulations; secondary transporters.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli
  • Genetic Vectors / genetics
  • Hydrogen-Ion Concentration
  • Methanocaldococcus / genetics*
  • Models, Molecular*
  • Molecular Dynamics Simulation
  • Protein Conformation
  • Sodium / metabolism*
  • Sodium-Calcium Exchanger / chemistry*
  • Sodium-Calcium Exchanger / genetics
  • Sodium-Calcium Exchanger / metabolism*
  • Thermodynamics


  • Sodium-Calcium Exchanger
  • Sodium