Architecture of the mitochondrial calcium uniporter

Abstract

Mitochondria from multiple, eukaryotic clades uptake and buffer large amounts of calcium (Ca2+) via an inner membrane transporter called the uniporter. Early studies demonstrated that this transport requires a mitochondrial membrane potential and that the uniporter is itself Ca2+ activated, and blocked by ruthenium red or Ru3601. Later, electrophysiological studies demonstrated that the uniporter is an ion channel with remarkably high conductance and selectivity2. Ca2+ entry into mitochondria is also known to activate the TCA cycle and appears to be critical for matching ATP production in mitochondria with its cytosolic demand3. MCU (mitochondrial calcium uniporter) is the pore forming and Ca2+ conducting subunit of the uniporter, but its primary sequence does not resemble any calcium channel known to date. Here, we report the structure of the core region of MCU, determined using nuclear magnetic resonance (NMR) and electron microscopy (EM). MCU is a homo-oligomer with the second transmembrane helix forming a hydrophilic pore across the membrane. The channel assembly represents a new solution of ion channel architecture and is stabilized by a coiled coil motif protruding in the mitochondrial matrix. The critical DxxE motif forms the pore entrance featuring two carboxylate rings, which appear to be the selectivity filter based on the ring dimensions and functional mutagenesis. To our knowledge, this is one of the largest structures characterized by NMR, which provides a structural blueprint for understanding the function of this channel.

Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter

“…It is not known whether Ru360 blocks Orai or CorA. Although previous functional studies established channel properties of MCU and the putative mechanism of Ru360 inhibition (5,6,14), structural information on ion and inhibitor binding is still missing.…”

“…The strong Mn 2+ binding is also consistent with the very slow Mn 2+ flux reported by previous patch-clamp experiments (5). The binding of Ru360 to the apex of the MCU pore has already been suggested based on the mutagenesis of S259 in human (6) and S238 in C. elegans (14). In the cMCU structure, S238 is adjacent to the D ring, and its hydroxyl group possibly interacts with the amine groups of Ru360 (Fig.…”

“…That structural study was performed on a Caenorhabditis elegans MCU construct with the N-terminal domain (NTD; residues 1-165) deleted, denoted as cMCU-ΔNTD. This protein construct has been shown to function as the full-length MCU in the mitochondrial Ca 2+ uptake assay (14). The MCU is a homopentamer with the second TM helix forming a hydrophilic pore across the presumed lipid bilayer.…”

“…In an earlier study, we determined the pore architecture of the MCU channel using structural data obtained from solution NMR and negative-stain EM experiments (14). That structural study was performed on a Caenorhabditis elegans MCU construct with the N-terminal domain (NTD; residues 1-165) deleted, denoted as cMCU-ΔNTD.…”

Strategic Positioning and Biased Activity of the Mitochondrial Calcium Uniporter in Cardiac Muscle

“…Without EMRE or MICU1, MCU has been shown to stay in a ϳ300-kDa complex (3) that might correspond to MCU-containing complexes from the mitochondrial fraction eluted in fractions 18 and 19. Considering the recently revealed pentameric structure, the MCU oligomer without any accessory proteins would be ϳ175 (5 ϫ 35) kDa (35). Because size exclusion fractions cannot be directly translated to molecular mass ranges, it would be difficult to tell if the end of the size range (fraction 20) could correspond to this pentamer.…”