Document Detail


Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria.
MedLine Citation:
PMID:  22495585     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV.
Authors:
Akos A Gerencser; Christos Chinopoulos; Matthew J Birket; Martin Jastroch; Cathy Vitelli; David G Nicholls; Martin D Brand
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-04-10
Journal Detail:
Title:  The Journal of physiology     Volume:  590     ISSN:  1469-7793     ISO Abbreviation:  J. Physiol. (Lond.)     Publication Date:  2012 Jun 
Date Detail:
Created Date:  2012-06-18     Completed Date:  2013-06-17     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  0266262     Medline TA:  J Physiol     Country:  England    
Other Details:
Languages:  eng     Pagination:  2845-71     Citation Subset:  IM    
Affiliation:
Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA 94945, USA. agerencser@buckinstitute.org
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MeSH Terms
Descriptor/Qualifier:
Animals
Calcium / metabolism
Cells, Cultured
Energy Metabolism
Fluorescent Dyes
Membrane Potential, Mitochondrial*
Models, Theoretical
Neurons / metabolism,  physiology*
Patch-Clamp Techniques
Rats
Rats, Inbred Strains
Rhodamines
Voltage-Sensitive Dye Imaging / methods*
Grant Support
ID/Acronym/Agency:
P01 AG025901/AG/NIA NIH HHS; PL1 AG032118/AG/NIA NIH HHS
Chemical
Reg. No./Substance:
0/Fluorescent Dyes; 0/Rhodamines; 0/tetramethylrhodamine methyl ester; 7440-70-2/Calcium
Comments/Corrections

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