on the exact molecular mechanism of MCU regulation. Our recent structural insight of the N-terminal domain revealed a b-grasp-like fold containing the MCU regulating acidic patch (MRAP) that binds Mg 2þ /Ca 2þ with mM affinity, destabilizes MCU, shifts self-association equilibrium to monomer and attenuates [Ca 2þ ] m uptake. The weak binding affinity for Mg 2þ is well suited for the regulation of MCU as mitochondrial matrix have higher Mg 2þ concentration (0.2-2 mM). Our recent identification of Mg 2þ as regulator of MCU channel, intrigued us to explore the molecular link between MCU and the unstudied Mg 2þ 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 Mg 2þ channel activity (I Mrs2p ) 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 I Mrs2p validating Mrs2p as an authentic mammalian mitochondrial Mg 2þ channel. Additionally, simultaneous measurement of [Ca 2þ ] m uptake and DJm was performed in cardiomyocytes isolated from WT and Mrs2p -/mice. In line with ablated I Mrs2p MCU-mediated Ca 2þ uptake was increased in Mrs2p -/myocytes, reinforcing the concept of Mg 2þ -dependent MCU regulation. Conversely, loss of MCU did not alter the I Mrs2p , suggesting a cation dependent MCU regulation that is consistent with other Ca 2þ channels including L-type, RyRs, IP 3 Rs, and CRAC. Mitochondrial ATP synthase has been shown recently to be vital not only for cellular energy production but also for energy dissipation and cell death. We identified and characterized a large non-selective uncoupling channel within the ATP synthase c-subunit ring, the persistent opening of which initiates cell death. We have continuing evidence for the crucial role of this channel in mitochondrial permeability transition (mPT). We have now purified ATP synthase from porcine heart mitochondria and performed single-channel studies. Excised proteoliposome patch-clamp recordings demonstrate that highly pure and fully assembled ATP synthase monomers form large conductance, Ca 2þ -sensitive and voltage-gated channels. We confirmed the monomeric state of ATP synthase by cryo-electron microscopy studies of ATP synthase reconstituted into proteoliposomes. We have also heterologously overexpressed and purified human ATP synthase c-subunit from E. coli plasma membranes. We show that human c-subunit purified from bacteria forms large conductance channels identical to those purified from HEK-293 cells. The channel is gated by polar amino acid residues situated at the mouth of the pore and by the hydrophilic F 1 portion of ATP synthase. We find that dissociation of ATP synthase F 1 from F O occurs when we expose primary hippocampal neurons to glutamate toxicity, suggesting that the non-reversible dissociation of F 1 from F O is pathological. We have successfully k...
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