The calcium (Ca 2+ ) uniporter of mitochondria is a holocomplex consisting of the Ca 2+ -conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca 2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn 2+ ), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn 2+ specifically, whereas in the WT the two rings bind Mn 2+ cooperatively, resulting in âŒ1,000-fold higher apparent affinity. Ca 2+ can specifically displace the bound Mn 2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn 2+ binding from the solventaccessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.) uptake by mitochondria is a long-known physiological phenomenon that is important for regulating various cellular activities such as aerobic metabolism and cell death (1-3). This uptake of Ca 2+ is achieved by a Ca 2+ uniporter whose activity can be blocked by ruthenium red (RuR) or ruthenium 360 (Ru360) (4). It was later shown by patch-clamping the inner mitochondrial membrane that this uniporter achieves remarkably high Ca 2+ conductance and selectivity (5). Only about 5 y ago was the molecular identity of the channel component of the uniporter identified using integrative genomics approaches (6, 7). This channel is commonly referred to as the mitochondrial calcium uniporter (MCU). In metazoans, however, the uniporter is much more complex than just the pore-forming subunit; it is a holocomplex (also called a uniplex) containing other regulatory components. Among them, the single-pass transmembrane (TM) protein EMRE (Essential MCU REgulator) is absolutely required for the MCU to conduct ions (8). Two soluble components in the intermembrane space, MICU1 and MICU2, are Ca 2+ -sensing proteins that gate the activity of MCU based on outside Ca 2+ concentrations through EMRE (9-12). The current consensus is that the minimum requirements for uniporter-mediated Ca 2+ flux in metazoans are MCU and EMRE, whereas in lower organisms such as Dictyostelium that lack EMRE, MCU i...