The mechanism of MICU-dependent gating of the mitochondrial Ca2+uniporter

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.


Sample-size estimation
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Replicates
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Replicate number of mitoplasts/cells/dishes is included in every figure. There were at least three biological replicates for all experiments. No points were excluded. All the required information is provided in the Figure Legends and Methods.

Statistical reporting
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Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: (For large datasets, or papers with a very large number of statistical tests, you may upload a single table file with tests, Ns, etc., with reference to sections in the manuscript.)

Group allocation
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No randomization was used. Samples were allocated according to the different KO cell lines or the cDNA constructs examined.
The source data is uploaded as Excel sheets labelled to indicate the corresponding Figures, and Raw Western blots images are provided as PDFs, for all the Figures and figure supplements. The code for image analysis is already uploaded on a server as indicated in the Methods section.