A Novel Ca2+ Release Channel in Mitochondria of Drosophila melanogaster: Properties and Role in Ca2+ Homeostasis

Mitochondrial Ca2+ uptake and release play a pivotal role in different physiological processes such as intracellular Ca2+ signaling and cell metabolism, while their dysregulation can lead to cell death induction. In energized mitochondria the Ca2+ uniporter (MCU) mediates Ca2+ uptake across the inner mitochondrial membrane (IMM) while the Na+/Ca2+ exchanger and the H+/Ca2+ exchanger are responsible for Ca2+ efflux. However, when mitochondrial matrix Ca2+ load exceeds the capacity of the efflux pathway by exchangers, an additional pathway for Ca2+ release from mitochondria may exist through opening of the permeability transition pore (PTP). The mitochondrial permeability transition (PT) describes a process of Ca2+-dependent, tightly regulated increase in the permeability of the IMM to solutes with molecular masses below 1500 Da, due to the opening of a high-conductance channel, the PTP. Prolonged PTP opening causes several effects, such as depolarization, osmotic swelling, outer mitochondrial membrane rupture and release of apoptogenic proteins like cytochrome c (cyt c). Transient openings of the PTP on the other hand, might be involved in physiological Ca2+ homeostasis and may protect mitochondria from Ca2+ overload. Several studies allowed a thorough characterization of the functional properties and regulation of the putative channel, but its molecular nature remains still elusive. One of the best defined modulators of the PTP is the mitochondrial peptidyl-prolyl-cis-trans-isomerase (PPIase) Cyclophilin D (Cyp-D), that plays an important role in protein folding and can be selectively inhibited by the immunosuppressant drug Cyclosporin A (CsA). In spite of its importance as a model organism and as a genetic tool, remarkably little is known about the properties of Ca2+ transport in mitochondria of the fruit fly Drosophila melanogaster, and on whether these mitochondria can undergo a PT. In this study we have characterized the pathways of Ca2+ transport in the digitonin-permeabilized embryonic Drosophila cell line S2R+. We demonstrated the presence of ruthenium red-sensitive Ca2+ uptake, and of Na+-stimulated Ca2+ release in energized mitochondria, which match well characterized Ca2+ transport pathways of mammalian mitochondria. Furthermore we identified and characterized a novel mitochondrial Ca2+-dependent Ca2+ release channel in Drosophila. Like the mammalian PTP, Drosophila Ca2+ release is inhibited by tetracaine and opens in response to matrix Ca2+ loading, IMM depolarization and thiol oxidation. As the yeast pore (and at variance from the mammalian PTP), the Drosophila channel is inhibited by Pi and insensitive to CsA. A striking difference between the pore of Drosophila and that of mammals is its selectivity to Ca2+ and H+ and the lack of mitochondrial swelling and cyt c release during the opening of the channel. The apparent absence of a mitochondrial Cyp in Drosophila prevents an investigation based on the effects of CsA, a classical inhibitor of the mammalian PTP. Thus, in the second part of this study we expressed human Cyp-D in Drosophila S2R+ cells in order to investigate its impact on the Ca2+-induced Ca2+ release channel. Preliminary Ca2+ retention capacity studies demonstrated that human Cyp-D induces opening of the Drosophila Ca2+ release channel in a rotenone-sensitive but CsA-insensitive manner. If the Cyp-D in Drosophila cells changes selectivity, size and properties of the Ca2+-induced Ca2+ release channel can now be addressed. We conclude that Drosophila mitochondria possess a selective Ca2+ release channel with features intermediate between yeast and mammals that is probably involved in Ca2+ homeostasis but not in Ca2+-mediated cell death induction. In this study we paved the way for the application from the sophisticated genetic strategies that Drosophila provides to define the molecular nature of the PTP and its role in pathophysiology of Ca2+ homeostasis