LAUSR

Magnesium (Mg 2+ ) is an important cation critical for cellular functions and tissue integrity. Mitochondria have been demonstrated to be capable of both accumulate and release Mg 2+ . However, the exact molecular machinery associated with mitochondrial Mg 2+ (mMg 2+ ) influx has not yet been delineated. In the present study we characterized the mammalian mMg 2+ channel, Mrs2 and comprehensively studied its role in energy metabolism. Protein flux, membrane fractionation and STED microscopy studies revealed Mrs2 to localize on the inner mitochondrial membrane with its N and C-terminus in the matrix. Western blot and qPCR analysis confirmed the ubiquitous distribution of Mrs2 in all metabolically active tissues. We adopted lentiviral based strategy to stably knock down (KD) Mrs2 in vitro . Primarily, the use of FRET-based mMg 2+ sensor, MitoMario showed a decreased influx of Mg 2+ into mitochondria in Mrs2 KD cells. This was further confirmed by patch clamping the mitoplasts of the control and Mrs2 KD cells. Because Mg 2+ is an important co-factor in the machineries that replicate, we next assessed the mitochondrial copy number. The decreased influx of mMg 2+ impacted the mitochondrial copy number and electron transport chain (ETC) complex assembly. The defective ETC assembly was marked by increased generation of mitochondrial reactive oxygen species, increased proton leak, decreased ATP levels, and also prompted a metabolic switch from mitochondrial oxidative phosphorylation to glucose oxidation in Mrs2 KD cells. Additionally, Mrs2 KD cells had an increased sensitivity to mROS-induced mitochondrial permeability transition pore opening. To further study the role of Mrs2 in cardiac mitochondrial metabolism and cellular energetics, we have successfully adopted the CRISPR/Cas9 mediated gene targeting strategy to generate the cardiac-specific Mrs2 knock out mouse model. Our study is the first of its kind to characterize the mitochondrial Mg 2+ channel and its impact on mitochondrial copy number and cell viability. Our findings not only identify Mrs2 as an authentic mitochondrial Mg 2+ channel, but also validates the critical role of mMg 2+ in maintaining the bioenergetic state of the cell.