Mechanistic Modeling of MCUx Gatekeeping, Ca2+ Transport, and Coupled Cytosolic-Mitochondrial Ca2+ Dynamics.

Mitochondrial Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUx), in which MICU1/2 serve as cytosolic Ca2+-sensing gatekeepers that set a Ca2+-dependent activation threshold. Coordinated control of MCUx activity is critical because mitochondrial Ca2+ uptake couples cytosolic Ca2+ signals to metabolic activation and must be regulated to prevent mitochondrial Ca2+ overload and bioenergetic dysfunction. We developed a mechanistic model that incorporates explicit MICU1/2-dependent MCUx gatekeeping and a thermodynamically constrained Ca2+ transport formulation that accounts for Mg2+ inhibition and membrane potential dependence. Cytosolic and mitochondrial Ca2+ dynamics were simulated by integrating the MCUx model into a mitochondrial cation-handling model under multiple Ca2+ stimulation protocols. Because matrix-side Ca2+ regulation of MCUx remains controversial, we also evaluated a putative matrix-side regulatory mechanism by comparing simulations with and without an added matrix-side regulatory module, rather than assuming such regulation as a required feature of the MCUx model. The model reproduces key experimental behaviors across genotypes. In wild-type mitochondria, MCUx-mediated Ca²⁺ uptake is negligible below a cytosolic Ca2+ threshold (~0.2 µM), whereas MICU1-knockout mitochondria show constitutive uptake and MICU2-knockout mitochondria exhibit an intermediate, lowered threshold. Inclusion of matrix-side MCUx regulation transiently attenuated MCUx-mediated Ca²⁺ uptake over an intermediate mitochondrial Ca2+ range, producing higher transient cytosolic Ca2+ and lower transient mitochondrial Ca2+, while both cases approached similar steady states. In addition, cytosolic Mg2+ acts as a graded inhibitor of MCUx-mediated Ca2+ uptake, limiting mitochondrial Ca2+ loading. These results provide a quantitative framework for coupled cytosolic-mitochondrial Ca²⁺ dynamics across diverse conditions.