Association Between β3-Adrenoceptor Activation and Intracellular Free Zinc Ion Increase Contributes to Hyperglycemia-Induced Cardiac ER-Stress

Abstract

Mitochondrial Ca 2þ regulates a wide variety of physiological processes, including ATP production and cell death. Rapid Ca 2þ uptake into mitochondria is mediated by the mitochondrial calcium uniporter (MCU) complex, which is composed of the pore forming MCU protein, and the regulatory proteins EMRE, and MICU1/MICU2. Currently, the submitochondrial localization and transmembrane orientation of these proteins are either unknown or under debate. We attack these issues using a classical thiol-modification approach. We removed native MCU complex proteins in HEK293 cells using CRISPR/Cas9, and reintroduced mutants containing a single cysteine at defined positions. Treatments of the mitoplast with a bulky, thiol-reactive compound polyethylene glycol (PEG) maleimide would alter the molecular weight of the protein if the engineered Cys is exposed to the intermembrane space. Using this strategy along with other biochemical methods, we demonstrated that (1) MCU adopts an orientation with the signature DIME motif facing intermembrane space (IMS), ( 2) the conserved C-terminal polyaspartic tail of EMRE is in the IMS, (3) both MICU1 and MICU2 are associated with the outer leaflet of the inner membrane, and (4) although MICU1 is a peripheral membrane protein, MICU2 is an integral component of the inner membrane. Then, applying domain interaction analysis and mutagenesis screening, we identified molecular contacts that govern the Ca 2þ transport behavior of the MCU complex. In particular, we demonstrate that EMRE interacts with MCU through the transmembrane helices to activate the Ca 2þ pore, while using the polyaspartic tail to recruit MICU1/2 to gate the pore. This dual functionality of EMRE ensures that all functional MCU complexes respond appropriately to Ca 2þ stimuli from the cytosol, safeguarding against dangerous Ca 2þ leakage, which could diminish mitochondrial energy output and potentially trigger apoptotic cell death.