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

Abstract

Fluorescent Ca2+ indicators have been essential for the analysis of Ca2+ signaling events within the cells of the CNS. However, a systematic evaluation of their potential adverse effects is lacking. Here we show that chemical Ca2+ indicators, but not a genetically encoded Ca2+ indicator, potently suppress the activity of Na+- and K+-dependent adenosine triphosphatase (Na,K-ATPase), independently from their Ca2+ chelating activity. Loading of commonly utilized Ca2+ indicators, including Fluo-4 acetoxymethyl (AM), Rhod-2 AM, Fura-2 AM and the Ca2+ chelator BAPTA AM, in cultures of neurons, astrocytes, cardiomyocytes and kidney proximal tubule epithelial cells suppressed Na,KATPase activity by 30–80%. Ca2+ indicators also suppressed agonist-induced activation of Na,K-ATPase, altered metabolic status and produced a dose-dependent loss of cell viability. In vivo, loading of Ca2+ indicators significantly altered brain extracellular concentrations of K+ and ATP. A critical review of the previous observations based on chemical Ca2+ indicators is therefore warranted.

Chemical Syntheses and Chemical Biology of Carboxyl Polyether Ionophores: Recent Highlights

“…[86] We suggest that by exchanging the ion sensor,t his or closely related assays could be used to visualize metal ion transport into vesicles by fluorescence microscopy in real time (Figure 2b). [88][89][90] Small-molecule ion sensors,although subject to constant improvements,are notoriously unselective between ions and can exert nonrelated secondary effects; [91] this calls for caution if they are employed for analysis of ion transport in cellular systems.Despite these challenges,highly selective ion sensors for, for example,C u + and Cu 2+ have been developed and rigorously characterized in cells and in vivo. [87] Metal ion sensors and their use in cells have been reviewed in detail.…”

“…[87] Metal ion sensors and their use in cells have been reviewed in detail. [88][89][90] Small-molecule ion sensors,although subject to constant improvements,are notoriously unselective between ions and can exert nonrelated secondary effects; [91] this calls for caution if they are employed for analysis of ion transport in cellular systems.Despite these challenges,highly selective ion sensors for, for example,C u + and Cu 2+ have been developed and rigorously characterized in cells and in vivo. [92] Fordetailed analyses of ionophore activities,however, subcellular specificity is required and although small molecules are easily targeted to active mitochondria by incorporation of lipophilic cations, [93] other organelles are more difficult to target by small molecules.I ft he ionophore perturbs the membrane potential of the mitochondria, this method for mitochondrial targeting might not be suitable.…”

Chemische Synthesen und chemische Biologie von Carboxylpolyether‐Ionophoren: Aktuelle Entwicklungen

“…[86] Wirs chlagen vor,d ass durch den Austausch des Ionensensors dieser oder eng verwandte Assays eingesetzt werden kçnnten, um den Metallionentransport in Vesikel durch Fluoreszenzmikroskopie in Echtzeit zu beobachten (Abbildung 2b). Niedermolekulare Ionensensoren sind, obwohl sie kontinuierlich optimiert werden, notorisch unselektiv fürIonen und kçnnen unzugehçrige Sekundäreffekte haben, [91] weshalb ihre Verwendung zur Analyse des Ionentransport in zellulären Systemen mit Vorsicht zu betrachten ist. Zur Analyse müssen die einzelnen Kompartimente beim Sampling direkt adressierbar sein, oder die Ionenaustauschreaktion muss schnell abgebrochen werden, damit die Proben zur Identifizierung und Quantifizierung der im Kompartiment abgefangenen Ionen prozessiert werden kçnnen;dies ist z.…”

“…[87] Metallionensensoren und deren Verwendung in Zellen wurden ausführlich in Übersichtsartikeln beschrieben. Niedermolekulare Ionensensoren sind, obwohl sie kontinuierlich optimiert werden, notorisch unselektiv fürIonen und kçnnen unzugehçrige Sekundäreffekte haben, [91] weshalb ihre Verwendung zur Analyse des Ionentransport in zellulären Systemen mit Vorsicht zu betrachten ist. Tr otz dieser Herausforderungen wurden hochselektive Ionensensoren, z.…”

Measurement of myofilament calcium in living cardiomyocytes using a targeted genetically encoded indicator

“…However, recent work has highlighted direct inhibition of the Na/K ATPase in many cell types by the BAPTA family of Ca ++ indicators 11 which limits cell survival and may reduce ATP dependent force production. Indeed fura2-based impairment of contractility has previously been observed in isolated rat cardiac trabeculae 12 .…”