NCLX, but not Letm1, mediates matrix Ca2+ extrusion and modulates the mitochondrial redox state during HeLa cell stimulation

Mitochondria sense and shape intracellular Ca2+ signals, acting as a cell signaling hub. The uptake of Ca2+ into the mitochondrial matrix activates intermediary and energy metabolism, and Ca2+ extrusion mechanisms ensure that this Ca2+ signal is transient. After a long quest, the proteins promoting Ca2+ uptake and release have been discovered: a mitochondrial Ca2+ uniporter was shown to mediate Ca2+ uptake, and two ion exchangers, NCLX and Letm1, were proposed to exchange Ca2+ against Na+ or H+ respectively.

To relate mitochondrial Ca2+ extrusion to mitochondrial function, we have manipulated the expression levels of NCLX and Letm1 and measured by single cell imaging their impact on matrix Ca2+, matrix redox state, and NAD(P)H concentration evoked by Ca2+ mobilizing agonists.

We find that the histamine stimulated mitochondrial Ca2+ rise is highly variable in individual HeLa cells. The rate of Ca2+ extrusion is a function of this amplitude, being highest for large matrix Ca2+ elevations but approaching 0 as the amplitudes decrease. We therefore propose a single-cell bi-parametric analysis that correlates the rate of matrix Ca2+ extrusion with the amount of Ca2+ uptake, to determine the impact of NCLX and Letm1. Over-expression of NCLX revealed enhanced mitochondrial Ca2+ extrusion in cells with initial high accumulation of Ca2+ into the matrix. On the other hand, expression (or depletion) of Letm1 had no impact on Ca2+ extrusion, at any level of matrix ion. Ca2+ has a dual effect on mitochondrial redox status, stimulating Ca2+-activated dehydrogenases (reducing) and accelerating respiration with associated enhanced formation of reactive oxygen (oxidizing). The histamine-induced Ca2+ rise increased the signal of the redox sensitive probe roGFP showing that this physiological Ca2+ rise causes a net reduction of the matrix. Enhancing Ca2+ extrusion following NCLX over-expression abolished the Ca2+ effect. Consistent with these findings, the matrix Ca2+ rise increased cellular NAD(P)H; an effect strongly reduced following NCLX over-expression. Pharmacological inhibition of mitochondrial Ca2+ extrusion, completely rescued both the reduced matrix redox status and NAD(P)H production in NCLX-expressing cells.

We conclude that the extrusion of mitochondrial matrix Ca2+ is mediated by NCLX but not by Letm1. By controlling the duration of matrix Ca2+ rises, NCLX contributes to the regulation of NAD(P)H production and modulates the mitochondrial redox state.