Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria

Abstract

Ca²⁺ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca²⁺ uptake machinery. The MCU is a Ca²⁺-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca²⁺ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca²⁺ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca²⁺ ionophore ETH129 significantly accelerated Ca²⁺ uptake. The residual Ca²⁺ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca²⁺ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca²⁺ uptake pathway in brain mitochondria that mediates residual Ca²⁺ influx and induction of PTP in a fraction of the mitochondrial population.

Keywords: calcium; cyclophilin D; energy metabolism; ion channel; mitochondria; mitochondrial membrane potential; mitochondrial permeability transition (MPT); respiration.