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. 2024 Jul;29(5):100166.
doi: 10.1016/j.slasd.2024.100166. Epub 2024 Jun 5.

Development of a live cell assay for the zinc transporter ZnT8

Lucia Azzollini  1 , Dolores Del Prete  2 , Gernot Wolf  3 , Christoph Klimek  3 , Mattia Saggioro  2 , Fernanda Ricci  2 , Eirini Christodoulaki  3 , Tabea Wiedmer  3 , Alvaro Ingles-Prieto  3 , Giulio Superti-Furga  4 , Lia Scarabottolo  2
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Free article

Development of a live cell assay for the zinc transporter ZnT8

Lucia Azzollini et al. SLAS Discov. 2024 Jul.
Free article

Abstract

Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic β-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic β-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.

Keywords: SLC; SLC30a8; Transport assay; Type 2 diabetes; Zinc transporter; ZnT.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lia Scarabottolo reports financial support was provided by Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777,372. RESOLUTE has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 777,372. This Joint Undertaking receives support from the European Union's Horizon 2020 research and innovation programme and EFPIA. This article reflects only the authors’ views and neither IMI nor the European Union and EFPIA are responsible for any use that may be made of the information contained therein. LA, DDP, MS, FR and LS are employed by Axxam s.p.a. GSF is a co-founder of the companies Proxygen and Solgate and a coauthor on patents related to SLCs. The Superti-Furga laboratory receives funds from Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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