Isolation of mitochondria and mitoplasts from tissues

Mice were sacrificed by CO₂ asphyxiation followed by cervical dislocation. For the preparation of mitoplasts from heart, SM, brown fat, kidney, and liver, the selected mouse tissues were isolated, rinsed, and homogenized in ice-cold medium containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, and 0.1% bovine serum albumin (BSA) (pH adjusted to 7.2 with Trizma® base) using a glass grinder with six slow strokes of a Teflon pestle rotating at 275 (soft tissues) or 600 (fibrous tissues) rotations per minute. The homogenate was centrifuged at 700 x g for 5–10 min to pellet nuclei and unbroken cells. For some tissues, the first nuclear pellet was resuspended in the same solution and homogenized again to increase the yield of mitochondria. Mitochondria were collected by centrifugation of the supernatant at 8,500 x g for 10 min.

Mitoplasts were produced from mitochondria using a French press. Briefly, mitochondria were suspended in a solution containing 140 mM sucrose, 440 mM D-mannitol, 5 mM HEPES, and 1 mM EGTA (pH adjusted to 7.2 with Trizma® base) and then subjected to a French press at 1200–2,000 psi to rupture the outer membrane. Mitoplasts were pelleted at 10,500 x g for 15 min and resuspended for storage in 500 μl of solution containing 750 mM KCl, 100 mM HEPES, and 1 mM EGTA (pH adjusted to 7.2 with Trizma® base). Mitochondria and mitoplasts were prepared at 0–4 °C and stored on ice for up to 5 h. Immediately before the electrophysiological experiments, 15–50 μl of the mitoplast suspension was added to 500 μl solution containing 150 mM KCl, 10 mM HEPES, and 1 mM EGTA (pH adjusted to 7.0 with Trizma® base) and plated on 5-mm coverslips pretreated with 0.1% gelatin to reduce mitoplast adhesion. Nearly all AAC molecules in isolated mitoplasts were initially in the c-state (see Fig. 1a and ​and3e).3e). A likely explanation for this initial c-state is the presence of endogenous adenine nucleotides in the matrix and not in the isolation media during mitochondria/mitoplast isolation.