Chabosseau Pauline, Tuncay Erkan, Meur Gargi, Bellomo Elisa A, Hessels Anne, Hughes Stephen, Johnson Paul R V, Bugliani Marco, Marchetti Piero, Turan Belma, Lyon Alexander R, Merkx Maarten, Rutter Guy A
Section of Cell Biology, Division of Medicine, and ‡National Heart and Lung Institute, Imperial College London , London, United Kingdom.
ACS Chem Biol. 2014 Sep 19;9(9):2111-20. doi: 10.1021/cb5004064. Epub 2014 Jul 17.
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.
锌（Zn2+）离子在细胞生理学中发挥重要作用的认识日益深入。虽然已确定胞质溶胶中的游离Zn2+浓度为0.1 - 1 nM，但亚细胞器中的游离Zn2+浓度尚未明确。在此，我们扩展了基因编码的荧光共振能量转移（FRET）锌离子探针的eCALWY家族，以实现对内质网（肌质网）（ER）和线粒体基质中锌离子的测量。将这些探针应用于多种哺乳动物细胞类型，结果显示静息状态下线粒体的游离[Zn2+]值约为300 pM，略低于胞质溶胶中的水平，但比最近使用另一种基于FRET的传感器所报道的值高3个数量级。相比之下，发现内质网中的游离[Zn2+]≥5 nM，这比最近报道的值高5000多倍，但与内质网作为可动员锌离子储存库的推测作用一致。用肌质网/内质网Ca2+ - ATP酶抑制剂处理β细胞或心肌细胞、用ATP进行嘌呤能刺激后内质网Ca2+的动员或内质网氧化还原的调控，均未对[Zn2+]ER产生可检测到的影响。这些发现对先前提出的Ca2+在内质网锌离子动员中的作用提出质疑，并表明内质网中高锌离子水平可能是细胞内稳态的一个重要方面。
