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

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.

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