Unveiling the K(+)-sensitivity of cell metabolism using genetically encoded, FRET-based K(+), glucose, and ATP biosensors

Investigating dynamic changes of mitochondrial ATP and cytosolic glucose levels of single living cells over time by genetically encoded biosensors provides an informative readout of their metabolic activities. Here, we describe how to monitor the metabolic K(+)-sensitivity of HEK293 cells exploiting ATP-, glucose-, and K(+) probes. Fluorescence live-cell imaging of these Förster resonance energy transfer-based biosensors over time in response to gramicidin, an ionophoric peptide, indicated an absolute dependency of cellular ATP homeostasis on high intracellular K(+) levels. For complete information on the generation and use of this protocol please refer to Bischof et al. (2021).