ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains

Abstract

Spatially and temporally controlled increases of H2O2 emerge as an intracellular signal. We hypothesized that ROS and Ca2+ interact locally, in the restricted volume of the ER-mitochondrial interface. These physically tethered structures host enrichments of ion transport proteins such as the IP3 receptor, which support elevated nanodomains of Ca2+ during signalling events and are sensitive to H2O2. We used the genetically encoded H2O2 sensor HyPer incorporated into an inducible linker system to probe the redox environment at the ER-mitochondrial interface in HepG2 cells. We found a moderately elevated H2O2 nanodomain which developes into a H2O2 transient following IP3 receptor-mediated ER Ca2+ release and mitochondrial Ca2+ uptake. Pharmacological inhibition showed that the transient was dependent upon ER Ca2+, mitochondrial membrane potential and functional electron transport chain. HyPer measurements of the mitochondrial intermembrane space revealed significantly elevated H2O2 within this volume. Using electron microscopy we found that HepG2 mitochondria possess a cohort of dilated cristae, which disappeared following IP3-linked Ca2+ release. Paxilline that inhibits mitochondrial BKCa channels blocked the cristae reshaping and also abolished the H2O2 transient at the interface. Furthermore, paxilline caused suppression of the IP3-linked calcium signal, whereas interface targeted killer red, a photoactivated H2O2 source, induced sensitization to the IP3-linked agonist. We conclude that the intermembrane/cristae volume of mitochondria represents an oxidized pool fed by the electron transport chain. Ca2+-uptake stimulates expansion of the mitochondrial matrix via K+ and concomitant water uptake, squeezing the oxidized volume of the cristae to the interface. Here, a transient H2O2 nanodomain sensitizes IP3 receptors to further stimulation. We demonstrate a physiological setting where Ca2+ release may autoregulate using mitochondrial H2O2 released from the cristae.