Respiratory Chain

Patch-clamp experiments using mitoplasts were performed as described previously [18] (link), [19] (link). Briefly, mitoplasts were prepared from a sample of human astrocytoma mitochondria placed in a hypotonic solution (5 mM HEPES, 200 µM CaCl₂, pH = 7.2) for approximately 1 min to induce swelling and breakage of the outer membrane. Then, a hypertonic solution (750 mM KCl, 30 mM HEPES, 200 µM CaCl₂, pH = 7.2) was added to restore the isotonicity of the medium. The patch-clamp pipette was filled with an isotonic solution containing 150 mM KCl, 10 mM HEPES, and 200 µM CaCl₂ at pH = 7.2. Mitoplasts are easily recognizable due to their size, round shape, transparency, and presence of a “cap”, characteristics that distinguish these structures from the cellular debris that is also present in the preparation. An isotonic solution containing 200 µM CaCl₂ was used as the control solution for all of the presented data. The low-calcium solution (1 µM CaCl₂) contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA and 0.752 mM CaCl₂ at pH = 7.2. All of the modulators of the channels and the substrates and inhibitors of the respiratory chain were added as dilutions in isotonic solution containing 200 µM CaCl₂. To apply these substances, we used a perfusion system containing a holder with a glass tube (made in our workshop), a peristaltic pump, and Teflon tubing. The mitoplasts at the tip of the measuring pipette were transferred into the openings of a multibarrel “sewer pipe” system in which their outer faces were rinsed with the test solutions (Fig. 1A). The configuration of our patch-clamp mode is presented in Fig. 1A. The experiments were carried out in patch-clamp inside-out mode. This is based on observations with various mitochondrial substrates applied such as NADH or succinate. Reported voltages are those applied to the patch-clamp pipette interior. Hence, positive potentials represent the physiological polarization of the inner mitochondrial membrane (outside positive).
The electrical connection was made using Ag/AgCl electrodes and an agar salt bridge (3 M KCl) as the ground electrode. The current was recorded using a patch-clamp amplifier (Axopatch 200B, Molecular Devices Corporation, USA). The pipettes, made of borosilicate glass, had a resistance of 10–20 MΩ and were pulled using a Flaming/Brown puller.
The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. The traces of the experiments were recorded in single-channel mode. The illustrated channel recordings are representative of the most frequently observed conductance for the given condition. The conductance of the channel was calculated from the current-voltage relationship (data not shown). The probability of channel opening (P_o, open probability) was determined using the single-channel search mode of the Clampfit 10.2 software. Calculations were performed using segments of continuous recordings lasting 60 s, with N>1000 events. Data from the experiments are reported as the mean values ± standard deviations (S.D.). Student’s t-test was used for statistical analysis. In figures showing single-channel recordings, “-” indicates the closed state of the channel.

Cellular oxidative phosphorylation and glycolysis alternations were determined with the Seahorse XF24 Flux Analyser (Seahorse Bioscience) by measuring extracellular acidification rates (ECAR) and oxygen consumption rates (OCR), respectively, in real time according to the manufacturer's instructions. HCC‐LM3 were seeded in a XF24‐well plate (Seahorse Bioscience) at a density of 3 × 10⁵ per well with ASPP2 knockdown cells and control cells, then allowed to attach overnight. OCR was assessed using sequential injection of 1 μM oligomycin, 1 μM carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP), 1 μM antimycin and rotenone. For assessment of ECAR, cells were incubated with unbuffered medium followed by injection of 10 mM glucose, 1 μM oligomycin (Sigma‐Aldrich) and 80 mM 2‐deoxyglucose (Sigma‐Aldrich). The basal levels of OCR and ECAR were recorded first, then the OCR and ECAR levels were recorded after sequential injection of the compounds that inhibit the respiratory mitochondrial election transport chain, ATP synthesis or glycolysis. Both OCR and ECAR measurements were normalized to cell numbers and reported as pmoles/min for OCR and mpH/min for ECAR.

All experimental XL-MS and CP-MS data for OGDHC from BHM presented in this manuscript were previously generated and are published by us. Detailed information about the sample preparation, cross-linking as well as CP can be found in the published manuscript entitled ‘Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain’ [35 (link)]. Raw data for XL-MS and CP-MS are publicly available at the ProteomeXchange partner PRoteomics IDEntifications (PRIDE) database and the ComplexomE profiling DAta Resource (CEDAR) database with the identifiers PXD025102 and CRX33, respectively.