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 Ca²⁺ ions, thereby sensing and shaping cellular Ca²⁺ signals. The Ca²⁺ uniporter MCU mediates Ca²⁺ uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca²⁺ against Na⁺ or H ⁺, respectively. Here we study the role of these ion exchangers in mitochondrial Ca²⁺ extrusion and in Ca²⁺-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca²⁺ efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca²⁺ ([Ca²⁺]_mt) elevations. NCLX overexpression enhanced the rates of Ca²⁺ efflux, whereas increasing LETM1 levels had no impact on Ca²⁺ 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⁺/Ca²⁺ 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⁺/Ca²⁺ exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca²⁺ 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 Ca²⁺ signals into redox changes.