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

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.