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

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• 作者：Pauline Chabosseau ; Erkan Tuncay ; Gargi Meur ; Elisa A. Bellomo ; Anne Hessels ; Stephen Hughes ; Paul R.V. Johnson ; Marco Bugliani ; Piero Marchetti ; Belma Turan ; Alexander R. Lyon ; Maarten Merkx ; Guy A. Rutter
• 刊名：ACS Chemical Biology
• 出版年：2014
• 出版时间：September 19, 2014
• 年：2014
• 卷：9
• 期：9
• 页码：2111-2120
• 全文大小：522K
• ISSN：1554-8937

文摘

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鈥? nM, the free Zn²⁺ concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded F枚rster 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 鈭?00 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 鈮? 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 尾-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.