Variable Assembly of EMRE and MCU Creates Functional Channels with Distinct Gatekeeping Profiles

Abstract

MCU is a Ca 2+ -selective channel that mediates mitochondrial Ca 2+ influx. The human channel contains tetrameric pore-forming MCU, regulatory subunits MICU1/2, and EMRE that is required both for channel function and MICU1/2-mediated Ca 2+ regulation. A structure of MCU with EMRE revealed a 4:4 stoichiometry, but the stoichiometry in vivo is unknown. Expression of tagged EMRE and MCU at a 1:10 ratio in cells lacking EMRE and MCU restored channel activity but not full channel gatekeeping. Increasing EMRE expression enhanced gatekeeping, raising the cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) threshold for channel activation. MCU-EMRE concatemers creating channels with 1EM-RE:4MCU restored Ca 2+ uptake in cells, whereas cells expressing concatemers that enforced a 4EM-RE:4MCU stoichiometry demonstrated enhanced channel gatekeeping. Concatemers enforcing 2EMRE/4MCU recapitulated the activity, gatekeeping, and size of endogenous channels. Thus, MCU does not require four EMRE, with most endogenous channels containing two, but complexes with 1-4 EMRE have activity with full or partial gatekeeping.