MEFs at 80% confluency were harvested, and mitochondria were isolated as previously described (31). Mitoplasts were spontaneously formed by diluting isolated mitochondria 100-fold in patching media (see below) for 10 min, which caused controlled swelling and rupture of the outer membrane. Mitoplasts can be easily distinguished from intact mitochondria, as they appear as larger and more translucent ring-like structures studded by a curled outer membrane. Inner-membrane patches were excised from mitoplasts after formation of a seal using micropipettes with approximately 0.3-μm tips and resistances of 10 to 30 megohms at room temperature. Patching medium was 150 mM KCl, 5 mM Hepes, 1.25 mM CaCl2, and 1 mM EGTA (pH 7.4). Voltages were clamped with a Dagan 1200 amplifier and reported as pipette potentials. Permeability was typically determined from stable current levels and/or total amplitude histograms of 30 s of data at +20 mV. pCLAMP version 8 (Axon Instruments) and WinEDR v2.3.3 (Strathclyde Electrophysiology Software) were used for current analysis as previously described (32). Sample rate was 5 kHz with 1- to 2-kHz filtration. To determine whether the large conductance activity was due to the MPTP through the ANT family, patching medium containing 1 mM ADP/MgCl2 was administered by bath perfusion.

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