Abstract
Ca(2+) entry into mitochondria is mediated by the Ca(2+) uniporter-channel complex containing MCU, the Ca(2+)-selective pore, and associated regulatory proteins. The roles of MICU proteins are controversial. MICU1 was proposed to be necessary for MCU activity, whereas subsequent studies suggested it inhibits the channel in the low-cytoplasmic Ca(2+) ([Ca(2+)](c)) regime, a mechanism referred to as "gatekeeping," that imposes a [Ca(2+)](c) threshold for channel activation at ∼1-3 μM. Here, we measured MCU activity over a wide range of quantitatively controlled and recorded [Ca(2+)](c). 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 Ca(2+) crosstalk between single inositol 1,4,5-trisphosphate receptor (InsP(3)R) and MCU channels.