Project Summary/Abstract The mitochondrial Ca2+ transport system modulates mitochondrial Ca2+ levels to control important cellular processes including ATP generation, cell-death pathways, and buffering of intracellular Ca2+ signals. Malfunction of mitochondrial Ca2+ transport induces cardiac ischemia-reperfusion injury and neurodegeneration, facilitates cancer metastasis, and provokes many other detrimental conditions in human disease. This system includes three major players, the mitochondrial Ca2+ uniporter complex, the Na+/Ca2+ exchanger (mediated by the NCLX protein), and the H+/Ca2+ exchanger (possibly mediated by Letm1). Although the mitochondrial Ca2+ uniporter has been studied extensively, the transport and regulatory mechanisms of the other two Ca2+ exchangers remain mostly unknown. These exchangers are important for physiology, because cardiac-specific deletion of NCLX causes heart failure, and loss of a copy of LETM1 in humans induces epilepsy in the deadly genetic disease Wolf-Hirschhorn syndrome. Here, we propose to study the fundamental mechanisms of these mitochondrial Ca2+ exchangers and their contribution to mitochondrial Ca2+ homeostasis. In Aim 1, we will determine the transmembrane topology and transport mechanisms of Letm1 using a wide range of methods, including functional analysis of liposome-reconstituted proteins, substituted cysteine accessibility scan, single- molecule photobleaching, and co-immunoprecipitation. Furthermore, we will employ new-generation CRISPR prime-editor tools to test the hypothesis that Letm1 is the protein that mediates mitochondrial H+/Ca2+ exchange and that it can load Ca2+ into mitochondria under physiological conditions. In Aim 2, we developed a novel procedure to purify human NCLX and reconstitute the protein in liposomes. This powerful tool will be employed to establish the Na+/Ca2+ exchange stoichiometry, Michaelis-Menten kinetic parameters, and the mechanisms underlying ion recognition. It will also allow us to determine how a small-molecule, membrane- permeant compound CGP-37157 potently inhibits NCLX, thus providing useful information to further improve this drug for potential clinical use. Completing the proposed work will fundamentally improve the scientific knowledge of two mitochondrial Ca2+ transport proteins that play important roles in human pathophysiology, and will pave the way for future endeavors to design new therapeutic strategies to treat debilitating diseases caused by abnormal mitochondrial Ca2+ transport and homeostasis.