Aequorin as a Useful Calcium-Sensing Reporter in .

In , calcium ions (Ca) regulate the activity of several signaling pathways, especially the calcineurin signaling pathway. Ca homeostasis is also important for cell polarization, hyphal extension, and plays a role in contact sensing. It is therefore important to obtain accurate tools with which Ca homeostasis can be addressed in this fungal pathogen. Aequorin from has been used in eukaryotic cells for detecting intracellular Ca. A codon-adapted aequorin Ca-sensing expression system was therefore designed for probing cytosolic Ca flux in . The availability of a novel water-soluble formulation of coelenterazine, which is required as a co-factor, made it possible to measure bioluminescence as a readout of intracellular Ca levels in . Alkaline stress resulted in an immediate influx of Ca from the extracellular medium. This increase was exacerbated in a mutant lacking the vacuolar Ca transporter , thus confirming its role in Ca homeostasis. Using mutants in components of a principal Ca channel (, ), the alkaline-dependent Ca spike was greatly reduced, thus highlighting the crucial role of this channel complex in Ca uptake and homeostasis. Exposure to the antiarrhythmic drug amiodarone, known to perturb Ca trafficking, resulted in increased cytoplasmic Ca within seconds that was abrogated by the chelation of Ca in the external medium. Ca import was also dependent on the Cch1/Mid1 Ca channel in amiodarone-exposed cells. In conclusion, the aequorin Ca sensing reporter developed here is an adequate tool with which Ca homeostasis can be investigated in .