FIGURE 8.

C203S-CypD expression in the liver mitochondria of CypD^−/− mice does not alter mitochondrial oxygen consumption and membrane potential. Liver mitochondrial samples were obtained as described in Fig. 7. Respiratory control ratio (RCR) (A) was determined using glutamate and malate as substrates by calculating state 3/state 2 using a Oxygraph electrode. In B, membrane potential was monitored using the fluorescent dye TMRE in coupled respiring mitochondria and then in the presence of 1 μm protonophore carbonyl cyanide p-chlorophenylhydrazone. In C, mitochondrial swelling assay was measured by absolute absorbance at baseline and after Ca²⁺ (250 μm) addition in the presence and absence of CsA in isolated liver mitochondria from each of the indicated groups as described in A. The data show the profile of absorbance at 540 nm of mitochondrial suspensions after exposure to 250 μm CaCl₂. The results represent average measurements from four independent preparations of liver mitochondria from separate mice. In D, CRC was measured using the calcium-sensitive probe calcium green-5N in the presence of 10 μm and 50 μm Ca²⁺ pulses. mCypD, mutant CypD.