Abstract
The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one- or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (KATP) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and KATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adenosine Diphosphate / analysis*
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Adenosine Diphosphate / biosynthesis
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Adenosine Triphosphate / analysis*
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Adenosine Triphosphate / biosynthesis
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Animals
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Archaeal Proteins / genetics
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Archaeal Proteins / metabolism
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Astrocytes / metabolism*
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Astrocytes / ultrastructure
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Biosensing Techniques*
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Cell Line
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Embryo, Mammalian
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Energy Metabolism / physiology
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Green Fluorescent Proteins / analysis
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Humans
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KATP Channels / genetics
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KATP Channels / metabolism
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Mice
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Microscopy, Fluorescence, Multiphoton
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Molecular Imaging
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Neurons / metabolism*
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Neurons / ultrastructure
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Primary Cell Culture
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Single-Cell Analysis
Substances
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Archaeal Proteins
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GlnK1 protein, Methanosarcina mazei
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KATP Channels
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Adenosine Diphosphate
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Adenosine Triphosphate