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. T... More
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 Mn import and export in mutant cells. While impaired Mn export was hypothesized given the essential role for SLC30A10 in cellular Mn export, impaired Mn 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 Mn 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.
