Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca
Aequorin
/ pharmacology
Calcium
/ metabolism
Calcium Channel Blockers
/ pharmacology
Calcium Channels
/ genetics
Drug Discovery
/ methods
Drug Evaluation, Preclinical
/ methods
Humans
Lactic Acid
/ pharmacology
Mitochondria
/ drug effects
Mitoxantrone
/ pharmacology
Saccharomyces cerevisiae
/ cytology
Aequorin
Calcium
Drug screening
Luminescence assay
Mitochondria
Mitochondrial calcium uniporter
Yeast
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2021
2021
Historique:
entrez:
3
6
2021
pubmed:
4
6
2021
medline:
10
7
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34080145
doi: 10.1007/978-1-0716-1270-5_5
doi:
Substances chimiques
Calcium Channel Blockers
0
Calcium Channels
0
SMDT1 protein, human
0
mitochondrial calcium uniporter
0
Lactic Acid
33X04XA5AT
Aequorin
50934-79-7
Mitoxantrone
BZ114NVM5P
Calcium
SY7Q814VUP
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
69-89Références
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