title: MCU encodes the pore conducting mitochondrial calcium currents.
abstract: Mitochondrial calcium (Ca(2+)) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca(2+) uniporter is a highly Ca(2+)-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 Ca(2+) 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 Ca(2+) channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca(2+) 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. DOI:http://dx.doi.org/10.7554/eLife.00704.001.
authors.affiliation: Cardiovascular Research Center , Massachusetts General Hospital , Boston , United States ; Department of Cardiology , Howard Hughes Medical Institute, Boston Children's Hospital , Boston , United States.
authors.forename: Dipayan
authors.initials: D
authors.lastname: Chaudhuri
authors.forename: Yasemin
authors.initials: Y
authors.lastname: Sancak
authors.forename: Vamsi K
authors.initials: VK
authors.lastname: Mootha
authors.forename: David E
authors.initials: DE
authors.lastname: Clapham
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; C034703:mitochondrial calcium uniporter; D002118:Calcium
keywords: Human; MCUR1; MICU1; calcium channel; electrophysiology; mitoplast; ruthenium red