The Molecular Basis for Rapid CA2+ Transport Across the Inner Mitochondrial Membrane

Ca2+ uptake by energized mitochondria regulates bioenergetics, apoptosis, and global Ca2+ signaling. Ca2+ entry into mitochondria is mediated by the Ca2+ uniporter-channel complex containing MCU, the Ca2+-selective pore, and associated regulatory proteins. The precise roles of these regulatory proteins and their relative stoichiometry in the complex have yet to be elucidated. MICU1 was proposed to be necessary for MCU activity whereas subsequent studies suggested a role for MICU1 and its paralog MICU2 in channel inhibition in the low-cytoplasmic Ca2+ ([Ca2+]c) regime, a mechanism referred to as “gatekeeping”. EMRE is required for MCU channel function and coupling of MICU1/2-mediated gatekeeping, but its stoichiometry relative to MCU in the native complex is unclear. We measured MCU activity over a wide range of quantitatively controlled and recorded [Ca2+]c to identify the regulatory function of the MICU1 and 2 and introduced mutations into Ca2+-binding sites in each protein to determine the mechanism by which [Ca2+]c controls channel activity. We then addressed the functional consequences of manipulating the relative stoichiometry of EMRE and MCU by introducing tagged MCU and EMRE in MCU/EMRE double-knockout cells to directly compare their expression and created a series of MCU-EMRE concatemers with multiple concatenated MCU subunits fused to EMRE, allowing us to “fix” their stoichiometry. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca2+ uptake to mitochondria localized to nanodomains of high (\u3e2 μM) [Ca2+]c. Furthermore, while incorporation of a single EMRE reconstitutes channel activity, additional EMRE subunits increase the [Ca2+]c-threshold required for relief of gatekeeping. Endogenous channels most likely contain two EMRE subunits which tether two MICU1/2-dimers to the complex. These findings have important implications for developing new therapies to target diseases arising from dysregulation of mitochondrial Ca2+-transport