Abstract
Mitochondrial membrane potential (MMP) plays a crucial role in the function of cells and organelles, involving various cellular physiological processes, including energy production, formation of reactive oxygen species (ROS), unfolded protein stress, and cell survival. Currently, there is a lack of genetically encoded fluorescence indicators (GEVIs) for MMP. In our screening of various GEVIs for their potential monitoring MMP, the Accelerated Sensor of Action Potentials (ASAP) demonstrated optimal performance in targeting mitochondria and sensitivity to depolarization in multiple cell types. However, mitochondrial ASAPs also displayed sensitivity to ROS in cardiomyocytes. Therefore, two ASAP mutants resistant to ROS were generated. A double mutant ASAP3-ST exhibited the highest voltage sensitivity but weaker fluorescence. Overall, four GEVIs capable of targeting mitochondria were obtained and named mitochondrial potential indicators 1-4 (MPI-1-4). In vivo, fiber photometry experiments utilizing MPI-2 revealed a mitochondrial depolarization during isoflurane-induced narcosis in the M2 cortex.
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