NCLX Protein, but Not LETM1, Mediates Mitochondrial Ca2+ Extrusion, Thereby Limiting Ca2+-induced NAD(P)H Production and Modulating Matrix Redox State

Umberto De Marchi,Jaime Santo-Domingo,Cyril Castelbou,Israel Sekler,Andreas Wiederkehr,Nicolas Demaurex

doi:10.1074/jbc.m113.540898

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.

Highlights

Whether mitochondrial Ca2ϩ extrusion is mediated by NCLX or LETM1 and controls matrix redox state is unknown
LETM1 did not alter Ca2ϩ efflux rates, regardless of the amplitude of [Ca2ϩ]mt elevations (Fig. 3, C–E). These data demonstrate that NCLX but not LETM1 levels limit the rates of Ca2ϩ extrusion from mitochondria, an effect most apparent during large [Ca2ϩ]mt elevations in HeLa cells that endogenously express both exchangers
Ca2ϩ sequestration in the mitochondrial matrix contributes to the buffering of cytosolic Ca2ϩ elevations and serves as a signal that activates mitochondrial Ca2ϩ-dependent processes [24, 25]

Summary

Background

Whether mitochondrial Ca2ϩ extrusion is mediated by NCLX (mitochondrial sodium/calcium exchanger) or LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) and controls matrix redox state is unknown. 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. Prolonged (pathological) accumulation of Ca2ϩ in the matrix space can lead to mitochondrial Ca2ϩ overload, followed by mitochondrial permeability transition pore opening [27,28,29], resulting in the activation of cell death signals [30, 31] To avoid this transition from stimulatory to detrimental effects of Ca2ϩ, mitochondria possess two membrane systems to extrude Ca2ϩ: the Naϩ/Ca2ϩ exchanger and the Hϩ/Ca2ϩ exchanger [5, 6]. We have assessed the importance of Ca2ϩ extrusion kinetics in the regulation of oxidative metabolism and in the control of the mitochondrial redox state

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION