title: Mitochondrial and ER-targeted eCALWY probes reveal high levels of free Zn2+.
abstract: 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.
authors.affiliation: Section of Cell Biology, Division of Medicine, and ‡National Heart and Lung Institute, Imperial College London , London, United Kingdom.
authors.forename: Pauline
authors.initials: P
authors.lastname: Chabosseau
authors.forename: Erkan
authors.initials: E
authors.lastname: Tuncay
authors.forename: Gargi
authors.initials: G
authors.lastname: Meur
authors.forename: Elisa A
authors.initials: EA
authors.lastname: Bellomo
authors.forename: Anne
authors.initials: A
authors.lastname: Hessels
authors.forename: Stephen
authors.initials: S
authors.lastname: Hughes
authors.forename: Paul R V
authors.initials: PR
authors.lastname: Johnson
authors.forename: Marco
authors.initials: M
authors.lastname: Bugliani
authors.forename: Piero
authors.initials: P
authors.lastname: Marchetti
authors.forename: Belma
authors.initials: B
authors.lastname: Turan
authors.forename: Alexander R
authors.initials: AR
authors.lastname: Lyon
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism*; D015335:Molecular Probes; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D007211:Indoles; D015335:Molecular Probes; D011994:Recombinant Proteins; D015032:Zinc; D002118:Calcium; C000543:cyclopiazonic acid