Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Poster Abstracts

18-POS Board 15 NEW EVIDENCES SUPPORTING THE OCCLUSION MECHANISM UNDERLYING MICU1 REGULATION OF THE MITOCHONDRIAL CALCIUM UNIPORTER Tsung Yun Liu 1,2 ; Ming-Feng Tsai 1,2 ; 1 University of Colorado Anschutz Medical Campus, Department of Physiology and Biophysics, Aurora, CO, USA 2 University of Virginia, molecular physiology and biological physics, Charlottesville, VA, USA The mitochondrial Ca2+ uniporter which is a Ca2+ channel that transports Ca2+ into mitochondria across the inner mitochondrial membrane (IMM), is a key player regulating intracellular Ca2+ signaling. It is known that the uniporter is regulated by a MICU1 subunit, but the exact mechanism is under debate. There are two opposite regulatory mechanisms. The occlusion model proposes that MICU1 blocks the uniporter’s calcium pathway when calcium concentration is low. When calcium concentration increases, MICU1 would then separate from the calcium pathway to open the channel. By contrast, the potentiation model argues that MICU1 never blocks, but potentiates the uniporter when calcium increases. To test these two models, we utilized a sodium-dependent IMM depolarization assay in WT versus MICU1-knockout (KO) HEK cells. After chelating divalent cations with EDTA, MICU1-KO cells exhibit significantly faster IMM depolarization. This result suggests that MICU1 blocks the uniporter when calcium concentration is low, thus providing support for the occlusion mechanism. We then investigated the effects of MICU1 depletion on uniporter activity in mouse embryonic fibroblast (MEF) cells. MICU1-KO led to reduced rate of mitochondrial calcium uptake, but we found that this is caused by decreased EMRE expression. When we fused EMRE to MCU to enforce an obligatory 1:1 MCU-EMRE stoichiometry, the uniporter exhibits similar activity in WT and MICU1-KO cells. These results suggest that MICU1 does not potentiate the uniporter. Altogether, our experiments advance our knowledge of uniporter regulation by providing new evidences supporting the occlusion model and refuting the potentiation mechanism.

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