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

James Hamilton,Tatiana Brustovetsky,Jacob E Rysted,Zhihong Lin,Yuriy M Usachev,Nickolay Brustovetsky

doi:10.1074/jbc.ra118.002926

James Hamilton, Tatiana Brustovetsky + Show 4 more

Open Access

https://doi.org/10.1074/jbc.ra118.002926

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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.

Highlights

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
All MCU-KO mice used in our experiments were genotyped (Fig. 1A) and all mitochondrial preparations were tested for MCU by immunoblotting (Fig. 1B)
We discovered a unique MCU-independent Ca2ϩ uptake mechanism existing in brain mitochondria isolated from MCU-KO mice (Figs. 1 and 9)

Summary

To whom correspondence should be addressed

Mitochondrial Ca2ϩ uptake and the ability of Ca2ϩ to regulate mitochondrial functions have been established for many years [1,2,3,4]. MCU KO failed to protect brain from hypoxic-ischemic damage, despite strongly hindering mitochondrial Ca2ϩ uptake and PTP induction [30] One reason for this controversy could be an incomplete inhibition of Ca2ϩ uptake in brain mitochondria from MCU-KO mice. Our results demonstrate that there is a unique MCU-independent, Ru360sensitive Ca2ϩ uptake in brain mitochondria that results in PTP induction in a fraction of vulnerable organelles. These findings evince a potential mechanism by which deletion of MCU might be not very efficacious in protecting brain mitochondria from PTP induction and, in protecting the brain from ischemia–reperfusion insults

Results

Discussion

Experimental procedures