Mitochondrial permeability transition in the crustacean Artemia franciscana: Absence of a Ca-regulated pore in the face of profound calcium storage

When mammalian mitochondria are exposed to high calcium and phosphate, a massive swelling, uncoupling of respiration and release of cytochrome c occur. These changes are mediated by opening of the mitochondrial permeability transition pore (MPTP). Activation of the MPTP in vivo in response to hypoxic and oxidative stress leads to necrotic and apoptotic cell death. Considering that embryos of the brine shrimp A. franciscana tolerate anoxia for years, we investigated the MPTP in this crustacean to reveal whether or not pore opening occurs. Minimum molecular constituents of the regulated MPTP in mammals are believed to be the voltage-dependent anion channel (VDAC), the adenine nucleotide translocators (ANTs) and cyclophilin D. Western blot analysis revealed that mitochondria from A. franciscana possess all three required components. When measured with a Ca-sensitive fluorescent probe, rat liver mitochondria release matrix calcium upon addition of ≥100 μM extra-mitochondrial Ca (MPTP opening), whereas brine shrimp mitochondria continue to take up extra-mitochondrial Ca and do not release internal stores even up to 1.0 mM exogenously added Ca (no MPTP opening). Furthermore, no swelling of A. franciscana mitochondria in response to added Ca was observed and no release of cytochrome c could be detected. HgCl2-dependent swelling and cytochrome c release were readily confirmed, which is consistent with the presence of an ’unregulated pore’. While the absence of a regulated MPTP in A. franciscana mitochondria could contribute to the extreme hypoxia tolerance in this species, we speculate that absence of the regulated MPTP may be a general feature of invertebrates.