Identification of Critical MCUR1 Domains in the Mitochondrial Calcium Uniporter Complex that Regulates Cellular Metabolism

Mitochondria are multivariate signal processors that decode diverse cellular inputs into various bioenergetic outputs. One such signal is the calcium (Ca 2+ ) flux between the cytosol and mitochondria which modulates the organelle’s function. Under physiologic cytosolic [Ca 2+ ], energy output (ATP) can be adjusted to meet cellular demands. To maintain the Ca 2+ homeostatic circuit, a selective mitochondrial Ca 2+ Uniporter (MCU) complex promotes Ca 2+ uptake. MCUR1 is an integral mitochondrial inner membrane protein which serves as a scaffold factor for the MCU complex and regulates its functional activity. The resolved NMR structure of the human MCUR1 protein revealed a high degree of conservation for 3 residues within the “head domain” of the protein, A170, L174, V203, and Y204. Objective We hypothesized that alterations of these critical residues would affect MCUR1 function, and thereby, MCU complex activity. Methods To test this hypothesis, MCU‐mediated mitochondrial calcium ( m Ca 2+ ) uptake, resting matrix m Ca 2+ levels, and mitochondrial bioenergetics such as NAD(P)H production and oxygen consumption rate (OCR) were evaluated. Results Remarkably, cells expressing the MCUR1 A170R/L174R mutant demonstrated near complete ablation of MCU‐mediated m Ca 2+ uptake, NAD(P)H production and OCR. Conversely, cells expressing MCUR WT , or the MCUR V203R/Y204R mutant demonstrated similar effects on MCU activity and mitochondrial bioenergetics suggesting a nominal change at the structural level. Conclusion Collectively, our results indicate that the MCUR1 head domain is necessary for proper MCU complex activity, and that MCU‐mediated m Ca 2+ uptake modulates mitochondrial bioenergetics and cell function. Support or Funding Information This research was funded by the National Institutes of Health R25R25DK078381 program. (NIHR01GM109882, R01HL086699, R01HL142673, DOD/CDMRP PR181598P and NIH1S10RR027327 to M. Madesh and R25DK078381 to W.B.R. B.T.E. is supported by the National Center For Advancing Translational Sciences of the National Institutes of Health under Award Number TL1TR002647. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.