The mitochondrial calcium uniporter transports Ca2+ via a ligand-relay mechanism.

The mitochondrial calcium uniporter (mtCU) is a multicomponent Ca2+-specific channel that imparts mitochondria with the capacity to sense the cytosolic calcium signals. The metazoan mtCU comprises the pore-forming subunit MCU and the essential regulator EMRE, arranged in a tetrameric channel complex, and the Ca2+ sensing peripheral proteins MICU1-3. The mechanism of mitochondrial Ca2+ uptake by mtCU and its regulation is poorly understood. Our analysis of MCU structure and sequence conservation, combined with molecular dynamics simulations, mutagenesis, and functional studies, led us to conclude that the Ca2+ conductance of MCU is driven by a ligand-relay mechanism, which depends on stochastic structural fluctuations in the conserved DxxE sequence. In the tetrameric structure of MCU, the four glutamate side chains of DxxE (the E-ring) chelate Ca2+ directly in a high-affinity complex (site 1), which blocks the channel. The four glutamates can also switch to a hydrogen bond-mediated interaction with an incoming hydrated Ca2+ transiently sequestered within the D-ring of DxxE (site 2), thus releasing the Ca2+ bound at site 1. This process depends critically on the structural flexibility of DxxE imparted by the adjacent invariant Pro residue. Our results suggest that the activity of the uniporter can be regulated through the modulation of local structural dynamics. A preliminary account of this work was presented at the 67th Annual Meeting of the Biophysical Society in San Diego, CA, February 18-22, 2023