Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca2+ at high concentrations. Here we describe a ratiometric low-affinity Ca2+ sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca2+ concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca2+ signals inside the organelle. The use of the sensor was demonstrated under three experimental paradigms: (1) ER Ca2+ oscillations in cultured astrocytes, (2) ex vivo functional mapping of cholinergic receptors triggering ER Ca2+ release in acute hippocampal slices from transgenic mice, and (3) in vivo sarcoplasmic reticulum Ca2+ dynamics in the muscle of transgenic flies. Our results provide proof of the suitability of the new biosensors to monitor Ca2+ dynamics inside intracellular organelles under physiological conditions and open an avenue to explore complex Ca2+ signaling in animal models of health and disease.
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