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

Abstract

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.