Measurements of Threshold of Mitochondrial Permeability Transition Pore Opening in Intact and Permeabilized Cells by Flash Photolysis of Caged Calcium

Changes in intracellular calcium concentration play a major role both in signal transduction and in cell death. In particular, mitochondrial Ca²⁺ overload is critically important as a determinant of irreversible cell injury. When accumulated above a threshold, matrix Ca²⁺ triggers opening of the mitochondrial permeability transition pore (mPTP), initiating ATP depletion and cell death via necrosis or by promoting cytochrome c release and initiating the apoptotic cascade. Measurement of mitochondrial Ca²⁺ uptake capacity (or the threshold for mPTP opening) is, therefore, important for understanding the mechanisms of pathophysiology in a variety of disease models and also for testing neuro- or cardioprotective drugs. We have, therefore, devised an approach that delivers Ca²⁺ directly to the matrix of mitochondria independently of uptake and therefore independently of potential (Δψ_m ) that allows direct study both of the Ca²⁺ efflux pathway and of the specific sensitivity of mPTP to Ca²⁺ . This is achieved using the photolytic release of Ca²⁺ by flash photolysis of caged Ca²⁺ using compounds, such as o -nitrophenyl EGTA, introduced into the cell as the acetoxymethyl (AM) ester (NP-EGTA, AM). This method can be used in both intact and permeabilized cells.