Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese

• Published in: The Journal of biological chemistry 2020-03, Vol.295 (12), p.3875-3890
• Main Authors: Horning, Kyle J., Joshi, Piyush, Nitin, Rachana, Balachandran, Rekha C., Yanko, Frank M., Kim, Kwangho, Christov, Plamen, Aschner, Michael, Sulikowski, Gary A., Weaver, C. David, Bowman, Aaron B.
• Format: Article
• Language: English
• Subjects: Animals; calcimycin; Calcimycin - chemistry; Calcimycin - pharmacology; Calcium - metabolism; calcium transport; Cell Line; Cell Survival - drug effects; Fura-2 - chemistry; HEK293 Cells; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - drug effects; Induced Pluripotent Stem Cells - metabolism; ionomycin; Ionomycin - chemistry; Ionomycin - pharmacology; ionophore; Ionophores - chemistry; Ionophores - pharmacology; Male; manganese; Manganese - analysis; Manganese - chemistry; Manganese - metabolism; Manganese - toxicity; Mass Spectrometry - methods; MESM; metal homeostasis; Methods and Resources; Mice; neurobiology; neurodegenerative disease; transport metal
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009781

Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma–MS, we validated that this assay, termed here “manganese-extracting small molecule estimation route” (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.