Molecular mechanisms of the mitochondrial calcium transport system - Project Summary/Abstract The mitochondrial Ca2+ transport system includes three members in the mitochondrial inner membrane: the mitochondrial calcium uniporter, the Na+/Ca2+ exchanger, and the H+/Ca2+ exchanger. These Ca2+ transport proteins work together to regulate mitochondrial Ca2+ homeostasis, oxidative phosphorylation, cell-death pathways, and cytoplasmic Ca2+ signaling. Abnormal mitochondrial Ca2+ transport can induce severe pathological conditions, such as ischemia-reperfusion injury, neurodegenerative disease, heart failure, and cancer metastasis. The goal of this project is to close critical knowledge gaps about the molecular mechanisms of these proteins. The mitochondrial calcium uniporter is a multi-subunit Ca2+ channel that conducts cytoplasmic Ca2+ into the mitochondrial matrix. Here, we will employ combined biochemical, biophysical, electrophysiological, and structural-biology methods to (1) establish the mechanisms by which a poorly-studied, neuron-specific MICU3 subunit potentiates the uniporter, (2) identify the functional roles of the MCUR1 subunit in the uniporter complex and characterize its molecular properties, and (3) determine how small-molecule compounds inhibit or activate the uniporter to unveil novel pharmacological mechanisms to manipulate uniporter activities. Mitochondrial Na+/Ca2+ and H+/Ca2+ exchange, mediated by NCLX and Letm1, respectively, export matrix Ca2+ back to the cytoplasm. The molecular basis of NCLX/Letm1 ion transport and regulation remains mostly unknown to date. We have now established liposome reconstitution and electrical-recording tools to subject these proteins to detailed functional scrutiny. Our aims include (1) establishing Michaelis-Menten kinetics of NCLX in a sided system, (2) investigating how NCLX can be pharmacologically inhibited and regulated by oxidation of conserved cysteines, and (3) testing an innovative hypothesis that Letm1 catalyzes H+/Ca2+ exchange using a similar antiport mechanism as CLC H+/Cl- exchangers. Completing this research will fundamentally improve our knowledge about the mechanisms governing substrate transport, regulation, and pharmacological modulation of the mitochondrial Ca2+ transport process. These efforts can provide mechanistic information guiding future endeavors to design new therapeutic strategies to treat diseases related to abnormal mitochondrial Ca2+ transport and homeostasis.
