The FASEB Journal express article 10.1096/fj.01-0828fje. Published online May 8, 2002. Release of mitochondrial Ca 2+ via the permeability transition activates endoplasmic reticulum Ca 2+ uptake David N. Bowser, Steven Petrou, Rekha G. Panchal, Megan L. Smart, and David A. Williams Department of Physiology, University of Melbourne, Victoria 3010 Australia Corresponding author: David A. Williams, Department of Physiology, University of Melbourne, Victoria 3010 Australia. E-mail: davidaw@unimelb.edu.au ABSTRACT Regulatory interactions between the endoplasmic reticulum (ER) and the mitochondria in the control of intracellular free Ca 2+ concentration ([Ca 2+ ] I ), may be of importance in the control of many cell functions, and particularly those involved in initiating cell death. We used targeted Ca 2+ sensors (cameleons) to investigate the movement of Ca 2+ between the ER and mitochondria of intact cells and focused on the role of the mitochondrial permeability transition (MPT) in this interaction. We hypothesized that release of Ca 2+ from mitochondria in response to a known MPT agonist (atractyloside) would cause release of ER Ca 2+ , perpetuating cellular Ca 2+ overload, and cell death. Targeted cameleons (mitochondria and ER) were imaged with confocal microscopy 23 days following transient transfection of human embryonic kidney 293 cells. Opening of the MPT resulted in specific loss of mitochondrial Ca 2+ (blocked by cyclosporin A), which was sequestered initially by ER. The ER subsequently released this Ca 2+ load, leading to a global Ca 2+ elevation, a response that was not observed when ER Ca 2+ -ATPases were blocked with cyclopiazonic acid. Thus, ER plays an important role in moderating changes in intracellular Ca 2+ following MPT and may play a key role in cell death initiated by mitochondrial mechanisms. Key words: mitochondrial permeability transition • targeted Ca 2+ sensors (cameleons) M itochondrial involvement in cell Ca 2+ signaling is now widely accepted. Mitochondria have the ability to sequester large amounts of Ca 2+ and tune cytosolic Ca 2+ events, including Ca 2+ transient duration and amplitude (1). More recently, investigations have focused on the close proximity of mitochondria with the primary intracellular Ca 2+ stores (endoplasmic reticulum, ER) and the involvement of mitochondrial Ca 2+ uptake mechanisms in tuning IP 3 R-mediated Ca 2+ responses (2). However, we have only a rudimentary understanding of the dynamics of mitochondrial Ca 2+ release via mitochondrial permeability transition pore (MPT) activation during cell death, and subsequent downstream effects on ER Ca 2+ stores. Two experimental strategies are commonly used to directly explore the Ca 2+ concentration in organelles, such as the mitochondria and ER of living, intact cells. The first involves loading of intact cells with membrane-permeant Ca 2+ -fluorophores, with subsequent quenching or removal of potentially confounding cytosolic fluorescence. This strategy has focused largely on the