Mitochondria are intracellular magnesium stores: Investigation by simultaneous fluorescent imagings in PC12 cells

To determine the nature of intracellular Mg²⁺ stores and Mg ²⁺ release mechanisms in differentiated PC12 cells, Mg²⁺ and Ca²⁺ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg²⁺ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg²⁺ concentration ([Mg²⁺]_i) and the [Ca ²⁺]_i in these cells. Possible candidates as intracellular Mg²⁺ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg²⁺]_i was induced by caffeine application, intracellular IP₃ or Ca²⁺ liberated by photolysis, it appears that no Mg²⁺ release mechanism thus exists that is mediated via the action of Ca²⁺ on membrane-bound receptors in the ER or via the offloading of Mg²⁺ from binding sites as a result of the increased [Ca²⁺]_i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg²⁺ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg²⁺]_i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg²⁺ store in PC12 cell. Simultaneous measurements of [Ca²⁺]_i and mitochondrial membrane potential, and also of [Ca²⁺]_i and [Mg ²⁺]_i, revealed that the initial rise in [Mg ²⁺]_i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg²⁺ in the FCCP-induced [Mg²⁺]_i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg²⁺ release.