Reconstructing calcium conductance in MCUb illuminates its molecular design for tuning the mitochondrial calcium uniporter

The mitochondrial Ca2+ uniporter mediates mitochondrial Ca2+ uptake to regulate cellular bioenergetics, Ca2+ signaling and survival, but excessive activity triggers Ca2+ overload and tissue injury. Cells counter this threat by expressing MCUb, a paralog of the uniporter’s pore-forming MCU subunit, to attenuate uniporter activity. Despite harboring the conserved Ca2+-coordinating DIME motif, MCUb paradoxically lacks conductance, a defining yet enigmatic feature underlying its uniporter-inhibitory role. Here, we demonstrate that MCUb’s non-conductivity stems from its inability to bind EMRE, a subunit essential for uniporter function, and that its N-terminal domain (NTD) exerts autoinhibition. Reinstating EMRE binding and relieving NTD-mediated inhibition rebuild Ca2+ conductance in MCUb, reaching ~80% of MCU activity. Wild-type MCUb exhibits ~30% of the inhibitory capacity of a pore-disrupting E249A variant, indicating that MCUb is a modest, rather than potent, negative regulator. These findings reveal how MCU-MCUb paralog divergence endows the uniporter with regulatory plasticity to fine-tune mitochondrial Ca2+ homeostasis. Cells prevent the mitochondrial Ca2+ uniporter from causing Ca2+ overload and cell death by expressing the non-conductive regulator MCUb. Here, the authors rebuild Ca2+ conductance in MCUb, revealing the molecular basis of its regulatory function.