NCLX protein, but not LETM1, mediates mitochondrial Ca2+ extrusion, thereby limiting Ca2+-induced NAD(P)H production and modulating matrix redox state

J Biol Chem. 2014 Jul 18;289(29):20377-85. doi: 10.1074/jbc.M113.540898. Epub 2014 Jun 4.

Abstract

Mitochondria capture and subsequently release Ca(2+) ions, thereby sensing and shaping cellular Ca(2+) signals. The Ca(2+) uniporter MCU mediates Ca(2+) uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca(2+) against Na(+) or H(+), respectively. Here we study the role of these ion exchangers in mitochondrial Ca(2+) extrusion and in Ca(2+)-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca(2+) efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca(2+) ([Ca(2+)]mt) elevations. NCLX overexpression enhanced the rates of Ca(2+) efflux, whereas increasing LETM1 levels had no impact on Ca(2+) extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca(2+)]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The [Ca(2+)]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na(+)/Ca(2+) exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca(2+) extrusion from mitochondria. By controlling the duration of matrix Ca(2+) elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca(2+) signals into redox changes.

Keywords: Calcium Imaging; Calcium Signaling; Mitochondria; Mitochondrial Transport; Redox Signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcium Signaling / physiology*
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Clonazepam / analogs & derivatives
  • Clonazepam / pharmacology
  • HeLa Cells
  • Humans
  • Kinetics
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • NAD / biosynthesis
  • NADP / biosynthesis
  • Oxidation-Reduction
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sodium-Calcium Exchanger / antagonists & inhibitors
  • Sodium-Calcium Exchanger / genetics
  • Sodium-Calcium Exchanger / metabolism*
  • Thiazepines / pharmacology

Substances

  • Calcium-Binding Proteins
  • LETM1 protein, human
  • Membrane Proteins
  • Mitochondrial Proteins
  • Recombinant Proteins
  • SLC8B1 protein, human
  • Sodium-Calcium Exchanger
  • Thiazepines
  • NAD
  • NADP
  • Clonazepam
  • CGP 37157