title: Measurements of threshold of mitochondrial permeability transition pore opening in intact and permeabilized cells by flash photolysis of caged calcium.
abstract: 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 (Δψm) 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.
authors.affiliation: Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK.
authors.forename: Andrey Y
authors.initials: AY
authors.lastname: Abramov
authors.forename: Michael R
authors.initials: MR
authors.lastname: Duchen
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D018613:Microscopy, Confocal; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D010782:Photolysis / Q000187:drug effects; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D012235:Rhodamines; C508966:o-nitrophenylegtazic acid acetoxymethyl ester; C401833:tetramethylrhodamine methyl ester; D004533:Egtazic Acid; D016572:Cyclosporine; D002118:Calcium