Calcium uniporter regulatory subunit MCUb, mitochondrial
negative regulation of calcium import into the mitochondrion
MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Stress.
<h4>Background</h4>The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca<sup>2+</sup> (<sub>m</sub>Ca<sup>2+</sup>) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology.<h4>Methods</h4>We created a stable <i>MCUB</i> knockout cell line (<i>MCUB</i><sup>-/-</sup>) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca<sup>2+</sup> exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex.<h4>Results</h4>Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing <sub>m</sub>Ca<sup>2+</sup> uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit <sub>m</sub>Ca<sup>2+</sup> overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in <sub>m</sub>Ca<sup>2+</sup> uptake impair mitochondrial energetics and contractile function.<h4>Conclusions</h4>We detail a new regulatory mechanism to modulate mtCU function and <sub>m</sub>Ca<sup>2+</sup> uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate <sub>m</sub>Ca<sup>2+</sup> uptake and cellular physiology.