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Calcium retention capacity assay evaluates inhibition of mitochondrial permeability transition pore

M. Awais, D. Latawiec, R. Sutton Liverpool Pancreatitis Research Group, Institute of Translational Medicine, University of Liverpool, UK 11/2017

Mitochondrial dysfunction is central to the pathogenesis of acute pancreatitis, ischemia-reperfusion injury of the heart, brain and kidney, muscular dystrophies and neurodegeneration. Mitochondrial dysfunction is the result of a sudden increase in permeability of the inner mitochondrial membrane (IMM), via persistent opening of a multi-protein channel known as the mitochondrial permeability transition pore (MPTP). This is followed by uncontrolled proton flow across the IMM and unregulated flux of water, ions and small solutes into and out of the mitochondrial matrix. This results in rupture of the outer mitochondrial membrane (OMM) and eventually cell death by necrosis. Therefore, MPTP is an attractive target for cell death prevention in a host of disease states.


The calcium retention capacity assay challenges isolated mitochondria with spikes of calcium ions. Upon opening of the MPTP, Ca2+ leaks into the assay buffer and increases fluorescence of the membrane-impermeable CalciumGreen™ dye. The Omega multi-mode plate reader has proven excellent robustness for performing the multiple injections as well as reliable fluorescent detection of the assay.

 

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