Fig. 5.

The mitochondrial Na⁺- or Li⁺-dependent Ca²⁺ exchange is mediated by NCLX. (A) Traces and rates of Na⁺-dependent mitochondrial Ca²⁺ efflux. Mitochondrial Ca²⁺ levels were recorded by monitoring RP-mt fluorescence in HEK-293 cells overexpressing either the human NCLX isoform (NCLX) or vector alone (control) and coexpressing RP-mt. Experiments were conducted on digitonin-permeabilized cells (see Materials and methods). Mitochondrial Ca²⁺ uptake was induced by superfusion with Na⁺-free solution (replaced by NMDG⁺) containing Ca²⁺ (60 μM). Ca²⁺ efflux was monitored following superfusion in Ca²⁺-free solution in the presence or absence of Na⁺. Note that the Ca²⁺ efflux was strictly Na⁺-dependent and was enhanced by the expression of NCLX (n = 8, **P < 0.01). (B) Traces and rates of Na⁺-dependent mitochondrial Na⁺ influx. Mitochondrial Na⁺ levels were measured in cells loaded with the Na⁺-sensitive dye CoroNa Red (see Materials and methods) as in A. Note the enhanced and reciprocal nature of the Na⁺ and Ca²⁺ transport rates, mediated by mitochondrial NCLX (n = 8, **P < 0.01). (C) Traces and rates of Na⁺-dependent mitochondrial Ca²⁺ efflux in cells transfected with either siNCLX or a scrambled siRNA (si control). The same experimental paradigm described in A was applied. Silencing of NCLX eliminated mitochondrial Na⁺-dependent Ca²⁺ efflux (n = 11, **P < 0.01). (D) Traces and rates of Li⁺-dependent mitochondrial Ca²⁺ efflux in cells transfected with either siNCLX or si control, using Li⁺-containing solution (replacing Na⁺; n = 11; *P < 0.05). Silencing of NCLX also diminished Li⁺-dependent Ca²⁺ efflux.