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Measurements of threshold of mitochondrial permeability transition pore opening in intact and permeabilized cells by flash photolysis of caged calcium.

Abramov, AY; Duchen, MR; (2011) Measurements of threshold of mitochondrial permeability transition pore opening in intact and permeabilized cells by flash photolysis of caged calcium. Methods Mol Biol , 793 pp. 299-309. 10.1007/978-1-61779-328-8_19.

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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.

Type: Article
Title: Measurements of threshold of mitochondrial permeability transition pore opening in intact and permeabilized cells by flash photolysis of caged calcium.
Location: United States
DOI: 10.1007/978-1-61779-328-8_19
Keywords: Animals, Calcium, Cyclosporine, Egtazic Acid, Ion Channel Gating, Membrane Potential, Mitochondrial, Mice, Microscopy, Confocal, Mitochondrial Membrane Transport Proteins, Neurons, Permeability, Photolysis, Rhodamines, Spectrometry, Fluorescence
UCL classification: UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Neurology
UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Neurology > Molecular Neuroscience
UCL > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Cell and Developmental Biology
URI: http://discovery.ucl.ac.uk/id/eprint/1326537
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