Biochemistry and molecular physiology of the mitochondrial calcium uniporter

ABSTRACT Mitochondria uptake Ca2+ via the uniporter, a Ca2+-activated Ca2+ channel within the inner membrane. The uniporter comprises the pore-forming subunit MCU, its paralog MCUb, a metazoan-specific regulator EMRE, and the EF-hand Ca2+-sensing proteins MICU1-3. Calcium stimulates mitochondrial ATP production by upregulating the TCA cycle dehydrogenases. However, excessive Ca2+ uptake can lead to the activation of apoptotic pathways. Hence the processes involved in mitochondrial calcium uptake and efflux, cumulatively referred to as the calcium cycle, must be precisely regulated. Recent studies have documented a broad spectrum of pathologies associated with impaired mitochondrial calcium cycle. The recessive loss of function mutations in MICU1 underlie an unusual metabolic myopathy and fatigue. Impaired uniporter function is a likely cause of cardiac and skeletal myopathies associated with Barth syndrome. Common single nucleotide polymorphism in MCU and MCUb is associated with cardiometabolic traits. MCU and MCUb expression is elevated in various forms of muscular dystrophy, while ablation of MCUb in macrophages leads to impaired skeletal muscle regeneration. A comprehensive understanding of the uniporter function and its role in a broad context of the mitochondrial calcium cycle is essential in specific muscle diseases, inflammation, and innate immunity. Hence, we plan to: (1) Reconstitute the mammalian Ca2+ uniporter holocomplex in proteoliposomes. A fully functional in vitro reconstitution system will allow detailed structure-function studies of Ca2+ transport and regulation of mammalian holocomplex, understand specific regulation by MICU1 splice variants such as MICU1.1, a muscle-specific isoform, and verify candidate chemical inhibitors that we have identified. (2) Characterize the function of MCUb, the paralog of MCU. The current dogma in the field is that MCUb is a non-conducting subunit of the uniporter that can exert dominant-negative activity. Our preliminary studies indicate an alternative possibility, which we will explore using both genetic loss of function studies, biochemistry, and functional reconstitution. (3) Define the mitochondrial calcium cycle. We have devised a high-throughput, pooled genetic screening technology called permeabilized-cell mitochondrial function sequencing (PMF-seq) to identify genes whose ablation positively or negatively impacts mitochondrial voltage response to Ca2+. We will now perform follow-up experiments on selected new genes we identified and perform additional sensitized genetic screens on the genetic knockout and chemical inhibitor backgrounds. These studies will yield a complete and detailed map of interdependencies between mitochondrial calcium cycle genes and their effectors.