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Publication Detail
Measurements of threshold of mitochondrial permeability transition pore opening in intact and permeabilized cells by flash photolysis of caged calcium.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Journal Article
  • Authors:
    Abramov AY, Duchen MR
  • Publication date:
    2011
  • Pagination:
    299, 309
  • Journal:
    Methods Mol Biol
  • Volume:
    793
  • Status:
    Published
  • Country:
    United States
  • Language:
    eng
  • Keywords:
    Animals, Calcium, Cyclosporine, Egtazic Acid, Ion Channel Gating, Membrane Potential, Mitochondrial, Mice, Microscopy, Confocal, Mitochondrial Membrane Transport Proteins, Mitochondrial Permeability Transition Pore, Neurons, Permeability, Photolysis, Rhodamines, Spectrometry, Fluorescence
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.
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