Integrating genetically encoded fluorescent sensors to elucidate the spatiotemporal choreography of necrosis by sodium overload

Necrosis by sodium overload (NECSO) is a distinct cell death modality induced by the chemical regulator necrocide 1 (NC1), which targets the transient receptor potential melastatin 4 (TRPM4) channel to drive excessive sodium influx and potassium efflux. This unique necrosis is characterized by the exchange of monovalent ions, a restrained ATP supply, redox disorder and a final membrane rupture as main features. Real-time monitoring of metabolic and redox causality during the whole process in living cells has been technically challenging. To bridge this gap, we integrate an advanced toolset of genetically encoded fluorescent sensors to monitor monovalent ions, energy metabolites, and redox equivalents with high spatiotemporal resolution. By directing these sensors to specific subcellular compartments, we successfully capture the real-time choreography of potassium loss specifically induced by NC1 via TRPM4. Furthermore, we establish a precise detection paradigm for evaluating energy currency by integrating sensors for NADH and ATP that are capable of subcellular imaging. We observe general and rapid NADH accumulation along with an ATP shortage in the mitochondria and cytosol. A concomitant reduction in mitochondrial oxidative stress is observed. This study not only elucidates the metabolic progression of a peculiar type of necrosis but also establishes a robust methodological framework for applying genetically encoded sensors to broader physiological and toxicological research.