Identification of EFHD1 as a novel Ca2+ sensor for mitoflash activation

•Identified four Ca2+-linked mitoflash activators (MICU1, SLC25A23, SLC25A25, EFHD1) and one inhibitor (LETM1).•Identified EFHD1 as a novel mitochondrial Ca2+ sensor for Ca2+-dependent mitoflash activation.•EF-hand domains of EFHD1 are required for its mitoflash-regulatory effects.

Mitochondrial flashes (mitoflashes) represent stochastic and discrete mitochondrial events that each comprises a burst of superoxide production accompanied by transient depolarization and matrix alkalinization in a respiratory mitochondrion. While mitochondrial Ca2+ is shown to be an important regulator of mitoflash activity, little is known about its specific mechanism of action. Here we sought to determine possible molecular players that mediate the Ca2+ regulation of mitoflashes by screening mitochondrial proteins containing the Ca2+-binding motifs. In silico analysis and targeted siRNA screening identified four mitoflash activators (MICU1, EFHD1, SLC25A23, SLC25A25) and one mitoflash inhibitor (LETM1) in terms of their ability to modulate mitoflash response to hyperosmotic stress. In particular, overexpression or down-regulation of EFHD1 enhanced or depressed mitoflash activation, respectively, under various conditions of mitochondrial Ca2+ elevations. Yet, it did not alter mitochondrial Ca2+ handling, mitochondrial respiration, or ROS-induced mitoflash production. Further, disruption of the two EF-hand motifs of EFHD1 abolished its potentiating effect on the mitoflash responses. These results indicate that EFHD1 functions as a novel mitochondrial Ca2+ sensor underlying Ca2+-dependent activation of mitoflashes.

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