Journal
ELIFE
Volume 2, Issue -, Pages -Publisher
ELIFE SCIENCES PUBLICATIONS LTD
DOI: 10.7554/eLife.00704
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Funding
- National Institutes of Health [F32HL107021, R24DK080261]
- Helen Hay Whitney Foundation
- Howard Hughes Medical Institute
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Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (I-MiCa), whereas overexpression increases it. Additionally, a classic feature of I-MiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel.
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