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

Kyle J. Horning, Piyush Joshi, Rachana Nitin, Rekha C. Balachandran, Frank M. Yanko, Kwangho Kim, Plamen Christov, Michael Aschner, Gary A. Sulikowski, C. David Weaver, Aaron B. Bowman, Mike Shipston

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3875-3890
Number of pages16
JournalJournal of Biological Chemistry
Volume295
Issue number12
DOIs
StatePublished - Mar 20 2020

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Horning, K. J., Joshi, P., Nitin, R., Balachandran, R. C., Yanko, F. M., Kim, K., Christov, P., Aschner, M., Sulikowski, G. A., David Weaver, C., Bowman, A. B., & Shipston, M. (2020). Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese. Journal of Biological Chemistry, 295(12), 3875-3890. https://doi.org/10.1074/jbc.RA119.009781