Calcium binding and translocation by the voltage-dependent anion channel: A possible regulatory mechanism in mitochondrial function

Mitochondria play a central role in energy metabolism, Ca²⁺ signalling, aging and cell death. To control cytosolic or mitochondrial Ca²⁺ concentration, mitochondria possess several Ca²⁺-transport systems across the inner membrane. However, the pathway for Ca²⁺ crossing the outer membrane has not been directly addressed. We report that purified voltage-dependent anion channel (VDAC) reconstituted into lipid bilayers or liposomes is highly permeable to Ca²⁺. VDAC contains Ca²⁺-binding sites that bind Ruthenium Red (RuR), La³⁺ and that RuR completely closed VDACs in single or multichannel experiments. Energized, freshly prepared mitochondria accumulate Ca²⁺ (500-700 nmol/mg of protein), and subsequently released it. The release of Ca²⁺ is accompanied by cyclosporin A-inhibited swelling, suggesting activation of permeability transition pore (PTP). RuR and ruthenium amine binuclear complex, when added to mitochondria after Ca²⁺ accumulation has reached a maximal level and before PTP is activated, prevented the release of Ca²⁺ and the accompanied mitochondrial swelling. RuR also prevented PTP opening promoted by atractyloside, an adenine nucleotide translocase inhibitor. These results suggest that VDAC, located in the mitochondrial outer membrane, controls Ca²⁺ transport into and from the mitochondria, and that the inhibition of Ca²⁺ uptake by RuR and La³⁺ may result from their interaction with VDAC Ca²⁺-binding sites. Inhibition of PTP opening or assembly by RuR and ruthenium amine binuclear complex suggest the involvement of VDAC in PTP activity and/or regulation. The permeability of VDAC to Ca²⁺ and its binding of Ca²⁺, suggest that VDAC has a role in regulation of the mitochondrial Ca²⁺ homoeostasis.