Fluorescent Ca(2+) indicators directly inhibit the Na,K-ATPase and disrupt cellular functions.

Fluorescent Ca(2+) indicators have been essential for the analysis of Ca(2+) signaling events in various cell types. We showed that chemical Ca(2+) indicators, but not a genetically encoded Ca(2+) indicator, potently suppressed the activity of Na(+)- and K(+)-dependent adenosine triphosphatase (Na,K-ATPase), independently of their Ca(2+) chelating activity. Loading of commonly used Ca(2+) indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM, and of the Ca(2+) 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%. Ca(2+) 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 Ca(2+) 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 Ca(2+) indicators may be necessary.