Figure 3. In isolated liver mitochondria from Ucp2^−/− mice, no ruthenium red (RR)-sensitive Ca²⁺ uniporter exists.

(a) Ca²⁺ uptake of suspended isolated liver mitochondria from wild-type (WT) animals was measured by monitoring the reduction of extra-mitochondrial Ca²⁺ on application of 140 nmol Ca²⁺ mg⁻¹ mitochondrial protein with Calcium Green-5N (0.1 μM). Experiments were performed in the absence (n = 7) and presence (WT + RR, n = 4; single asterisk indicates P <0.05 versus WT) of ruthenium red, an established inhibitor of mitochondrial Ca²⁺ uniporter (100 nM). (b) In suspended liver mitochondria isolated from Ucp2^−/− mice, Ca²⁺ uptake (measured as described in a), was reduced (Ucp2^−/−; n = 7). The remaining Ca²⁺ uptake was insensitive to ruthenium red (100 nM; Ucp2^−/− + RR; n = 4). n.s. versus Ucp2^−/−. (c) Kinetics of mitochondrial Ca²⁺ uptake within 30 s after addition of Ca²⁺ calculated from the data presented in a and b. The asterisk indicates P <0.05 versus WT. (d) Fluorescence image of fura-2 loaded single mitochondria freshly isolated from mouse liver. The scale bar represents 10 μm. (e) In isolated single liver mitochondria from wild-type animals, mitochondrial Ca²⁺ sequestration was measured in the absence (WT, n = 44) and presence (WT + RR, n = 22) of ruthenium red (1 μM) by mitochondrial fura-2. The asterisk indicates P <0.05 versus WT. (f) Ca²⁺ uptake in isolated single mitochondria from Ucp2^−/− mice measured by mitochondrial fura-2 was reduced (Ucp2^−/−, n = 34) compared with that obtained in the wild-type littermates. The remaining Ca²⁺ uptake into the liver mitochondria of Ucp2^−/− mice was not ruthenium red-sensitive (Ucp2^−/− + RR, n = 21). n.s. versus Ucp2^−/−. (g) Ruthenium red-sensitive mitochondrial Ca²⁺ uptake in single mitochondria freshly isolated from wild-type and Ucp2^−/− (Ucp2^−/−, asterisk indicates P <0.05 versus WT) mice calculated from data in e and f. The error bars represent s.e.m (a–f) or indicate 95% confidencence intervals at the times shown (g).