Abstract
Manganese (Mn), an essential metal, can be toxic at elevated levels. In 2012, the first inherited cause of Mn excess was reported in patients with mutations in SLC30A10, a Mn efflux transporter. To explore the function of SLC30A10 in vitro, the current study used CRISPR/Cas9 gene editing to develop a stable SLC30A10 mutant Hep3B hepatoma cell line and collagenase perfusion in live mice to isolate primary hepatocytes deficient in Slc30a10. We also compared phenotypes of primary vs. non-primary cell lines to determine if they both serve as reliable in vitro models for the known physiological roles of SLC30A10. Mutant SLC30A10 Hep3B cells had increased Mn levels and decreased viability when exposed to excess Mn. Transport studies indicated a reduction of 54Mn import and export in mutant cells. While impaired 54Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired 54Mn import was unexpected. Whole genome sequencing did not identify any additional mutations in known Mn transporters in the mutant Hep3B mutant cell line. We then evaluated 54Mn transport in primary hepatocytes cultures isolated from genetically altered mice with varying liver Mn levels. Based on results from these experiments, we suggest that the effects of SLC30A10 deficiency on Mn homeostasis can be interrogated in vitro but only in specific types of cell lines.
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Data availability
Authors are willing to share cell lines, datasets, and protocols employed. Further information and requests for resources should be directed to lead contact at thomas_bartnikas@brown.edu.
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Acknowledgments
We acknowledge: Dr. Mitchell Knutson, University of Florida for providing Slc39a14 knockout mice who originally obtained from Drs Hojyo and Fukada; Dr. Christoph Schorl and the Genomics Core for assistance with RNA analyses (this facility has received partial support from the National Institutes of Health (Grant Nos. NIGMS P30GM103410, NCRR P30RR031153, P20RR018728, and S10RR02763), National Science Foundation (Grant No. EPSCoR 0554548), Lifespan Rhode Island Hospital, and the Division of Biology and Medicine, Brown University); Drs. Joseph Orchardo and David Murray for assistance with metal analysis; and Dr. Jennifer Sanders for assistance with primary hepatocyte isolation.
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Research was supported by National Institute of Health grants DK84122 and DK110049 (TBB).
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Conceptualization: TBB, MP; Methodology: TBB, MP; Formal analysis and investigation: MP, MAP, CJM, HLC; Writing—original draft preparation: MP; Writing—review and editing: TBB, MP; SH, TF: provided Slc39a14 mutant mouse strain; Funding acquisition and resources: TBB; Supervision: TBB.
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Prajapati, M., Pettiglio, M.A., Conboy, H.L. et al. Characterization of in vitro models of SLC30A10 deficiency. Biometals 34, 573–588 (2021). https://doi.org/10.1007/s10534-021-00296-y
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DOI: https://doi.org/10.1007/s10534-021-00296-y