Activity-based CO₂ sensing using CarboSenR2 provides new insights into cellular metabolism

Carbon dioxide (CO₂) is an ancient and ubiquitous physiological gas that is produced during aerobic respiration, consumed during photosynthesis and is present in the Earth's atmosphere at steadily increasing levels in modern history. CO₂ has often been considered a simple waste product of metabolism and has to date garnered considerably less research activity compared to that of oxygen, the substrate of aerobic respiration. However, recent research has demonstrated important roles of CO₂ in immunometabolism, immunology, skeletal and smooth muscle physiology, epithelial cell behaviour, cellular signalling and clinical medicine. Identification of CO₂ dependent post-translational modifications using recently developed mass spectrometric approaches has directly linked CO₂ to protein function (independent of CO₂ -associated changes in pH) strengthening the argument for further research in this area. Notably, there has been a lack of reliable tools to directly monitor CO₂ in living systems to date. CarboSenR2 is a new CO₂ selective fluorescent molecular sensor which has not been fully evaluated in vitro and has not been specifically applied to study CO₂ production in cellula. Here, we demonstrate the utility of CarboSenR2 as an activity-based CO₂ sensor in multiple cell systems using flow cytometric and microscopy based approaches. These data demonstrate that CarboSenR2 is sensitive to CO₂ concentrations within the physiological and pathophysiological range observed in humans and reveal the intriguing presence of mitochondrial-associated R-Dye microdomains within cells. Thus, these findings highlight the potential of CarboSenR2 to facilitate new investigations into the role and dynamics of CO₂ in physiological systems.