Evidence supporting the MICU1 occlusion mechanism and against the potentiation model in the mitochondrial calcium uniporter complex

• Published in: Proceedings of the National Academy of Sciences - PNAS 2023-04, Vol.120 (16), p.e2217665120-e2217665120
• Main Authors: Tsai, Chen-Wei, Liu, Tsung-Yun, Chao, Fan-Yi, Tu, Yung-Chi, Rodriguez, Madison X, Van Keuren, Anna M, Ma, Zhiwei, Bankston, John, Tsai, Ming-Feng
• Format: Article
• Language: English
• Subjects: Allosteric properties; Apoptosis; Bioenergetics; Biological Sciences; Blocking; Calcium; Calcium (intracellular); Calcium (mitochondrial); Calcium - metabolism; Calcium Channels - metabolism; Calcium currents; Calcium ions; Calcium signalling; Calcium, Dietary; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Depolarization; EmrE protein; Imports; Intracellular signalling; Membrane potential; Mitochondria; Mitochondrial Membrane Transport Proteins - genetics; Mitochondrial Membrane Transport Proteins - metabolism; Mitochondrial Membranes - metabolism; Molecular modelling; Occlusion; Pore formation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.2217665120

The mitochondrial calcium uniporter is a Ca channel that imports cytoplasmic Ca into the mitochondrial matrix to regulate cell bioenergetics, intracellular Ca signaling, and apoptosis. The uniporter contains the pore-forming MCU subunit, an auxiliary EMRE protein, and the regulatory MICU1/MICU2 subunits. Structural and biochemical studies have suggested that MICU1 gates MCU by blocking/unblocking the pore. However, mitoplast patch-clamp experiments argue that MICU1 does not block, but instead potentiates MCU via allosteric mechanisms. Here, we address this direct clash of the proposed MICU1 function. Supporting the MICU1-occlusion mechanism, patch-clamp demonstrates that purified MICU1 strongly suppresses MCU Ca currents, and this inhibition is abolished by mutating the MCU-interacting K126 residue. Moreover, a membrane-depolarization assay shows that MICU1 prevents MCU-mediated Na flux into intact mitochondria under Ca -free conditions. Examining the observations underlying the potentiation model, we found that MICU1 occlusion was not detected in mitoplasts not because MICU1 cannot block, but because MICU1 dissociates from the uniporter complex. Furthermore, MICU1 depletion reduces uniporter transport not because MICU1 can potentiate MCU, but because EMRE is down-regulated. These results firmly establish the molecular mechanisms underlying the physiologically crucial process of uniporter regulation by MICU1.