Abstract Body: The mitochondrial calcium uniporter mediates Ca²⁺ entry into the matrix, and matches energetic supply to demand during cardiac activity. However, many cardiac diseases are characterized by mitochondrial Ca²⁺ overload, such as ischemia/reperfusion (I/R) injury. The uniporter channel is typically formed by a tetramer of MCU subunits. These subunits have two transmembrane domains and create a pore lined with negative charges to allow rapid and selective Ca²⁺ permeation. Intriguingly, many studies have shown that a close homolog, MCUB can potentially oligomerize with MCU subunits to alter the pore. When the tetramer is made of MCU Ca²⁺ transmission is facilitated, but the presence of MCUB inhibits Ca²⁺ intake through the channel, suggesting that it is a dominant negative subunit. Moreover, recent studies in animals subject to ischemic cardiovascular injury has revealed endogenous upregulation of the MCUB subunit, proposed as an endogenous mechanism to prevent Ca²⁺ overload. Deletion of MCUB enhances activation and uptake when exposed to high Ca²⁺, making MCUB^−/− cells prone to mitochondrial Ca²⁺ overload and pore opening. Here, we investigate the mechanisms by which MCUB regulates the behavior of the channel, focusing on how MCUB may influence complex composition and regulate its targeting. For this study, we performed structure-function studies of reconstituted MCUB in MCU^-/- MCUB^-/- double knockout HEK 293T cells. Using modified MCUB constructs and a range of MCU to MCUB transfection ratios, we queried how many MCUB subunits can successfully incorporate into uniporter complex. We subsequently created a range of tetrameric concatemers featuring different ratios of MCU to MCUB, allowing us to observe that increasing amounts of MCUB incorporation lead to greater inhibition of mitochondrial Ca²⁺ uptake. Finally, we examined whether replacing portions of MCUB with the corresponding MCU sequence was able to rescue Ca²⁺ uptake, finding that these substitutions failed to significantly increase mitochondrial Ca²⁺ uptake. Our findings clarify how MCUB incorporation into the uniporter complex leads to dominant-negative inhibition of mitochondrial Ca²⁺ uptake.