Published January 1, 2014
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Mitochondrial and ER-Targeted eCALWY Probes Reveal High Levels of Free Zn2+
Creators
- 1. Univ London Imperial Coll Sci Technol & Med, Div Med, Sect Cell Biol, London, England
- 2. Univ Oxford, Nuffield Dept Surg Sci, Oxford OX3 9DU, England
- 3. Univ Pisa, Dept Endocrinol & Metab, Pisa, Italy
- 4. Ankara Univ, Fac Med, Dept Biophys, TR-06100 Ankara, Turkey
Description
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 Forster Resonance Energy Transfer (FRET) Zn2+ probes to permit measurements in the endo(sarco)plasmic reticulum (ER) and mitochondria! matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free [Zn2+] values of similar to 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 beta-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.
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