Project Summary The mitochondrial calcium uniporter complex (MCUcx) is a highly-selective, tetrameric Ca2+ channel that plays a primary role in transporting Ca2+ from the cytoplasm into the mitochondrial matrix. Although Ca2+ uptake by mitochondria plays a crucial role in stimulating ATP production, matrix Ca2+ overload can trigger opening of the mitochondrial permeability transition pore, leading to cell death. MCU dysfunction has been implicated in several pathological conditions such as heart failure, ischemia–reperfusion injury, neurodegeneration, cancer and skeletal muscle dystrophies. Recent biochemical and structural studies have shown that the MCUcx is a macromolecular complex composed of a pore-forming MCU subunit, an essential subunit EMRE, and EF-hand domain-containing MICU1–3 subunits. How each subunit of the MCU complex comes together and regulates mitochondrial Ca2+ entry has been the focus of many recent studies. Using direct mitochondrial patch-clamp methodology and an innovative heterologous expression system, we recently performed a comprehensive structure–function analysis of the MCUcx in the inner mitochondrial membrane. This expression system consists of gene knockout of each individual subunit of the MCUcx (more than 6 different knockout lines were generated by CRISPR/cas9) and is readily amenable to different patch-clamp configurations with a high success rate. Using this system and the patch-clamp technique, we propose to answer central questions in the field: 1) Where is the MCU gate, and how is this gate regulated by Ca2+ and MCU accessory subunits? and 2) How do the unique features in the MCU macromolecular structures (as revealed by recent structural studies) relate to MCU gating and function? Accomplishment of the aims of this proposal will provide better understanding of the mechanisms that regulate Ca2+ entry via the MCU and create an essential framework for the development of pharmacological interventions that target MCU gating for therapeutic purposes.