Research ArticleCell Biology

Fluorescent Ca2+ indicators directly inhibit the Na,K-ATPase and disrupt cellular functions

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Science Signaling  30 Jan 2018:
Vol. 11, Issue 515, eaal2039
DOI: 10.1126/scisignal.aal2039

The dark side of tracking Ca2+ in cells

Ca2+ signaling events in many different cell types are tracked with fluorescent Ca2+ indicators, such as Fluo-4, Rhod-2, and Fura-2, and can be inhibited with the Ca2+ chelator BAPTA. Smith et al. found that these commonly used reagents inhibited the Na,K-ATPase, a membrane protein that exchanges intracellular Na+ for extracellular K+ and thus helps set the resting membrane potential and regulate cellular volume. This inhibition, which was accompanied by reduced cell viability, decreased glucose uptake, and cell swelling, occurred in multiple cell types, including neurons, astrocytes, and cardiomyocytes, and in mice when Rhod-2 or Fluo-4 was microdialyzed into the CNS. However, a genetically encoded Ca2+ indicator did not inhibit the Na,K-ATPase. These results suggest that it may be necessary to use these reagents with caution or rely on genetically encoded indicators to prevent cellular toxicity from affecting experimental outcomes.


Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events in various cell types. We showed that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppressed the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca2+ chelating activity. Loading of commonly used Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca2+ chelator BAPTA AM into cultured mouse or human neurons, astrocytes, cardiomyocytes, or kidney proximal tubule epithelial cells suppressed Na,K-ATPase activity by 30 to 80%. Ca2+ indicators also suppressed the agonist-induced activation of the Na,K-ATPase, altered metabolic status, and caused a dose-dependent loss of cell viability. Loading of Ca2+ indicators into mice, which is carried out for two-photon imaging, markedly altered brain extracellular concentrations of K+ and ATP. These results suggest that a critical review of data obtained with chemical Ca2+ indicators may be necessary.

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