Mitochondrial and ER-Targeted eCALWY Probes Reveal High Levels of Free Zn²⁺

Zinc (Zn²⁺) ions are increasingly recognized as playing an important role in cellular physiology. Whereas the free Zn²⁺ concentration in the cytosol has been established to be 0.1–1 nM, the free Zn²⁺ concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded Förster Resonance Energy Transfer (FRET) Zn²⁺ probes to permit measurements in the endo­(sarco)­plasmic reticulum (ER) and mitochondrial matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free [Zn²⁺] values of ∼300 pM, somewhat lower than in the cytosol but 3 orders of magnitude higher than recently reported using an alternative FRET-based sensor. By contrast, free ER [Zn²⁺] was found to be ≥5 nM, which is >5000-fold higher than recently reported but consistent with the proposed role of the ER as a mobilizable Zn²⁺ store. Treatment of β-cells or cardiomyocytes with sarco­(endo)­plasmic reticulum Ca²⁺-ATPase inhibitors, mobilization of ER Ca²⁺ after purinergic stimulation with ATP, or manipulation of ER redox, exerted no detectable effects on [Zn²⁺]_ER. These findings question the previously proposed role of Ca²⁺ in Zn²⁺ mobilization from the ER and suggest that high ER Zn²⁺ levels may be an important aspect of cellular homeostasis.