Mitochondrial Respiration Under a Permeabilized-Cell Assay
Digitonin-permeabilized HeLa cells are suspended in a respirometer at 37 °C. You add pyruvate plus malate (state 2), then ADP (state 3), then oligomycin, then FCCP, then antimycin A. Oxygen consumption rates (pmol O2 s−1 per 10^6 cells) are: 12, 38, 9, 55, and 3. In a parallel run using succinate plus rotenone, the corresponding rates are 15, 42, 10, 60, and 4. A colleague says the FCCP step proves that the cells are making more ATP because oxygen consumption rises. Another student claims the antimycin A rate represents non-mitochondrial oxygen consumption. You also have a cell line with a heterozygous frameshift in the gene encoding the adenine nucleotide translocase (ANT). The lab asks whether the data support the colleague’s claim and whether the ANT mutant should be used to test the role of the proton gradient.
1. Calculate the respiratory control ratio (state 3/state 2) for each substrate and interpret the effects of oligomycin, FCCP, and antimycin A. Does the data support the colleague’s claim that FCCP increases ATP synthesis? Explain.
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RCR = state 3/state 2: pyruvate/malate 38/12 = 3.2; succinate 42/15 = 2.8. Both are coupled. Oligomycin inhibits ATP synthase, so respiration falls to 9 and 10, close to state 2, showing most oxygen consumption was driving ATP synthesis. FCCP dissipates the proton gradient, so oxygen consumption rises to 55 and 60, but ATP synthesis falls because the gradient is bypassed; increased respiration is not evidence of more ATP. Antimycin A blocks complex III, leaving 3–4 units of non-mitochondrial oxygen consumption. Therefore the colleague’s claim is wrong: FCCP increases uncoupled respiration, not ATP output.
2. Predict the effect of adding atractyloside, an ANT inhibitor, during state 3 with pyruvate/malate. Would the ANT heterozygous mutant be a suitable tool to test whether the FCCP effect depends on the proton gradient? Justify.
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Atractyloside blocks the adenine nucleotide translocase, preventing ADP uptake and ATP export. During state 3, ATP synthesis stops, so respiration should fall toward state 2 (but not to the antimycin A level) as the proton gradient builds. A heterozygous ANT frameshift is unlikely to abolish ANT function; haploinsufficiency may be partial, so it is a poor tool to test the role of the proton gradient. Better tools are ATP synthase inhibitors, uncouplers, and direct measurements of ΔΨ or ΔpH. If the mutant is used, compare homozygous knockout or rescue, and measure both respiration and ATP synthesis.