A 40 kDa protein of the inner membrane is the mitochondrial calcium uniporter

Mitochondrial Ca(2+) homeostasis plays a key role in the regulation of aerobic metabolism and cell survival(1), but the molecular identity of the Ca(2+) channel, the mitochondrial calcium uniporter(2), was still unknown. We have identified in silico a protein (denominated MCU) that shares tissue distribution with MICU1, a recently characterized uniporter regulator(3), coexists with uniporter activity in phylogeny and includes two trasmembrane domains in the sequence. siRNA silencing of MCU in HeLa cells drastically reduced mitochondrial Ca(2+) uptake. MCU overexpression doubled the [Ca(2+)](mt) rise evoked by IP(3)-generating agonists, thus significantly buffering the cytosolic elevation. The purified MCU protein exhibited channel activity in planar lipid bilayers, with electrophysiological properties and inhibitor sensitivity of the uniporter. A mutant MCU, in which two negatively-charged residues of the putative pore forming region were replaced, had no channel activity and reduced agonist-dependent [Ca(2+)](mt) transients when overexpressed in HeLa cells. Overall, these data demonstrate that the identified 40 kDa protein is the channel responsible for Ruthenium Red-sensitive mitochondrial Ca(2+) uptake, thus providing molecular basis for this process of utmost physiological and pathological relevance.