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Updated: Nov 3, 2025
Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells
Published on: April 27, 2018
Drug Discovery Assay to Identify Modulators of the Mitochondrial Ca2+ Uniporter
Daniela M Arduino1, Valerie Goh2, Dejana Mokranjac3
1Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum München and German National Diabetes Center (DZD), Neuherberg, Germany. daniela.moniz@helmholtz-muenchen.de.
Abstract:
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.
Insights
We developed a drug screening protocol to find inhibitors of the mitochondrial calcium uniporter (MCU), a key regulator of cell functions and a therapeutic target. This assay uses engineered yeast mitochondria for robust and sensitive identification of MCU-targeting drugs.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- The mitochondrial calcium uniporter (MCU) regulates cellular calcium homeostasis, impacting metabolism, signaling, and cell death.
- Dysfunctional MCU activity is implicated in various human diseases, positioning it as a significant therapeutic target.
- Developing specific MCU inhibitors is crucial for potential disease interventions.
Purpose of the Study:
- To establish a robust and sensitive drug screening protocol for identifying specific inhibitors of the mitochondrial calcium uniporter (MCU).
- To utilize engineered yeast mitochondria as a screening reagent for MCU activity.
- To differentiate direct MCU inhibition from secondary effects in drug discovery.
Main Methods:
- Employing cryopreserved yeast mitochondria engineered to express human MCU and its regulator EMRE, along with the aequorin luminescence calcium sensor.
- Utilizing D-lactate as an energy substrate in a mannitol/sucrose buffer to energize mitochondria and facilitate calcium uptake measurements.
- Implementing a screening assay designed for sensitivity, robustness, and ease of laboratory implementation to minimize false discovery rates.
Main Results:
- Successfully established a functional screening assay based on reconstituted MCU-mediated calcium uptake in yeast mitochondria.
- Demonstrated the ability to discriminate between direct and secondary drug effects on mitochondrial calcium uptake.
- Validated the assay's sensitivity and robustness for identifying MCU inhibitors.
Conclusions:
- The developed protocol provides a reliable method for screening MCU-targeting drug candidates.
- This assay facilitates the discovery of novel therapeutics for diseases associated with MCU dysfunction.
- The methodology is adaptable for widespread use in drug discovery laboratories.
