Mitochondrial and ER-targeted eCALWY probes reveal high levels of free Zn2+.
Mitochondrial and ER-targeted eCALWY probes reveal high levels of free Zn2+.
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DOI：
10.1021/cb5004064
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发表时间：
2014-09-19
影响因子：
4
通讯作者：
Rutter GA
中科院分区：
文献类型：
--
作者：
Chabosseau P;Tuncay E;Meur G;Bellomo EA;Hessels A;Hughes S;Johnson PR;Bugliani M;Marchetti P;Turan B;Lyon AR;Merkx M;Rutter GA
锌离子（Zn2+）在细胞生理中起着重要的作用。细胞质溶胶中的游离Zn2+浓度已确定为0.1-1 nM，而亚细胞器中的游离Zn2+浓度尚未确定。在这里，我们扩展了遗传编码Förster共振能量转移（FRET） Zn2+探针的eCALWY家族，以允许在内质网（sarco）和线粒体基质中进行测量。在一系列哺乳动物细胞类型中，这些探针显示静止线粒体游离[Zn2+]值为~ 300 pM，略低于细胞质，但比最近报道的使用另一种基于fret的传感器高出三个数量级。相比之下，发现游离ER [Zn2+]≥5 nM，比最近报道的高5000倍，但与ER作为可移动Zn2+存储的作用一致。用sarco（endo）质网Ca2+-ATP酶抑制剂治疗β细胞或心肌细胞，在ATP嘌呤能刺激后动员ER Ca2+，或操纵ER氧化还原，对[Zn2+]ER没有可检测到的影响。这些发现质疑了之前提出的Ca2+在内质网Zn2+动员中的作用，并表明高内质网Zn2+水平可能是细胞稳态的一个重要方面。
Zinc (Zn2+) ions are increasingly recognised 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 range of mammalian cell types, these probes reveal resting mitochondrial free [Zn2+] values of ~ 300 pM, somewhat lower than in the cytosol but three 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 mobilisable 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.
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影响因子：
9.3
作者：
Maret, Wolfgang
通讯作者：
Maret, Wolfgang
影响因子：
4
作者：
Bozym, Rebecca A.;Thompson, Richard B.;Fierke, Carol A.
通讯作者：
Fierke, Carol A.
影响因子：
5.4
作者：
Kaltenberg, Jennifer;Plum, Laura M.;Haase, Hajo
通讯作者：
Haase, Hajo
影响因子：
4.8
作者：
Brown, AM;Kristal, BS;Cooper, AJL
通讯作者：
Cooper, AJL
影响因子：
4.8
作者：
LINK, TA;VONJAGOW, G
通讯作者：
VONJAGOW, G