Article

Respective Contribution of Mitochondrial Superoxide and pH to Mt-cpYFP Flash Activity.

University of Rochester Medical Center, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 03/2013; DOI: 10.1074/jbc.M113.455709
Source: PubMed

ABSTRACT Superoxide flashes are transient bursts of superoxide production within the mitochondrial matrix that are detected using the superoxide sensitive biosensor, mitochondrial-targeted circularly permuted YFP (mt-cpYFP). However, due to the pH sensitivity of mt-cpYFP, flashes were suggested to reflect transient events of mitochondrial alkalinization. Here, we simultaneously monitored flashes with mt-cpYFP and mitochondrial pH with carboxy-SNARF-1. In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio (corresponding to a pH increase of <0.1) indicating that matrix alkalinization is minimal during a mt-cpYFP flash. Individual flashes were also accompanied by stepwise increases of MitoSOX signal and decreases of NADH autofluorescence, supporting the superoxide origin of mt-cpYFP flashes. Transient matrix alkalinization induced by NH4Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner. Low concentrations of nigericin (< 100nM) that resulted in a mild dissipation of the mitochondrial pH gradient increased flash frequency, while a maximal concentration of nigericin (5 μM) collapsed the pH gradient and abolished flash activity. These results indicate that mt-cpYFP flash events reflect a burst in ETC-dependent superoxide production that is coincident with a modest increase in matrix pH. Furthermore, flash activity depends strongly on a combination of mitochondrial oxidation and pH gradient.

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Jun 28, 2014