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Arduino, D.M. ; Goh, V. ; Mokranjac, D.* ; Perocchi, F.

Drug discovery assay to identify modulators of the mitochondrial Ca2+ uniporter.

In: Mitochondrial Medicine. Berlin [u.a.]: Springer, 2021. 69-89 (Methods Mol. Biol. ; 2277)
The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.
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Publication type Article: Edited volume or book chapter
Keywords Aequorin ; Calcium ; Drug Screening ; Luminescence Assay ; Mitochondria ; Mitochondrial Calcium Uniporter ; Yeast
ISSN (print) / ISBN 1064-3745
e-ISSN 1940-6029
Book Volume Title Mitochondrial Medicine
Quellenangaben Volume: 2277, Issue: , Pages: 69-89 Article Number: , Supplement: ,
Publisher Springer
Publishing Place Berlin [u.a.]
Reviewing status Peer reviewed