Molecular Link Between Mcu And Mrs2p Channels For Mitochondrial Ion Homeostasis And Energy Metabolism

Ca2+ has been shown to modulate mitochondrial metabolism through the activation of Ca2+-dependent dehydrogenases. [Ca2+]m uptake is facilitated by the large electrochemical gradient across the inner mitochondrial membrane and mediated by the Mitochondrial Calcium Uniporter (MCU). MCU is a hetero-oligomeric complex and known to be regulated by several of its interacting partners MICUs, MCUR1, EMRE and MCUb. But there is a lack of knowledge on the exact molecular mechanism of MCU regulation. Our recent structural insight of the N-terminal domain revealed a β-grasp-like fold containing the MCU regulating acidic patch (MRAP) that binds Mg2+/Ca2+ with ∼mM affinity, destabilizes MCU, shifts self-association equilibrium to monomer and attenuates [Ca2+]m uptake. The weak binding affinity for Mg2+ is well suited for the regulation of MCU as mitochondrial matrix have higher Mg2+ concentration (0.2-2 mM). Our recent identification of Mg2+ as regulator of MCU channel, intrigued us to explore the molecular link between MCU and the unstudied Mg2+ selective transporter, Mrs2p for mitochondrial ion homeostasis and bioenergetics. To establish the link between MCU and Mrs2p, we generated a CRISPR/Cas9-mediated Mrs2p global knockout mouse model. Mitochondrial Mg2+ channel activity (IMrs2p) was measured in mitoplast obtained from cardiomyocytes isolated from WT and Mrs2p-/- mice by adopting our well devised patch-clamp experiments. Knocking out Mrs2p significantly ablated IMrs2p validating Mrs2p as an authentic mammalian mitochondrial Mg2+ channel. Additionally, simultaneous measurement of [Ca2+]m uptake and ΔΨm was performed in cardiomyocytes isolated from WT and Mrs2p-/- mice. In line with ablated IMrs2p MCU-mediated Ca2+ uptake was increased in Mrs2p-/- myocytes, reinforcing the concept of Mg2+-dependent MCU regulation. Conversely, loss of MCU did not alter the IMrs2p, suggesting a cation dependent MCU regulation that is consistent with other Ca2+ channels including L-type, RyRs, IP3Rs, and CRAC.