The store-operated calcium (Ca
) channel Orai governs Ca
influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca
stored in ...the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of
Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca
selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.
The calcium release-activated calcium channel Orai regulates Ca
entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca
release from ...the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on
Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.
The mitochondrial calcium uniporter is a Ca2+-gated ion channel complex that controls mitochondrial Ca2+ entry and regulates cell metabolism. MCU and EMRE form the channel while Ca2+-dependent ...regulation is conferred by MICU1 and MICU2 through an enigmatic process. We present a cryo-EM structure of an MCU-EMRE-MICU1-MICU2 holocomplex comprising MCU and EMRE subunits from the beetle Tribolium castaneum in complex with a human MICU1-MICU2 heterodimer at 3.3 Å resolution. With analogy to how neuronal channels are blocked by protein toxins, a uniporter interaction domain on MICU1 binds to a channel receptor site comprising MCU and EMRE subunits to inhibit ion flow under resting Ca2+ conditions. A Ca2+-bound structure of MICU1-MICU2 at 3.1 Å resolution indicates how Ca2+-dependent changes enable dynamic response to cytosolic Ca2+ signals.
The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca2+ channel of the inner ...mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU–EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 Å resolution. Using a novel assay, we demonstrate robust Ca2+ uptake into proteoliposomes containing the purified complex. Uptake is dependent on EMRE and also on the mitochondrial lipid cardiolipin. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU–EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.
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•MCU and EMRE are components of the mitochondrial Ca2+ uniporter ion channel.•Using a novel assay, we interrogate Ca2+ uptake by purified MCU–EMRE.•A cryo-EM structure of the MCU–EMRE complex is determined at 3.5 Å resolution.•The structure has noteworthy differences from a human MCU–EMRE structure.•The structures may represent alternate conformations.
The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel and a major route of calcium entry into mitochondria. How the channel catalyses ion permeation and achieves ...ion selectivity are not well understood, partly because MCU is thought to have a distinct architecture in comparison to other cellular channels. Here we report cryo-electron microscopy reconstructions of MCU channels from zebrafish and Cyphellophora europaea at 8.5 Å and 3.2 Å resolutions, respectively. In contrast to a previous report of pentameric stoichiometry for MCU, both channels are tetramers. The atomic model of C. europaea MCU shows that a conserved WDXXEP signature sequence forms the selectivity filter, in which calcium ions are arranged in single file. Coiled-coil legs connect the pore to N-terminal domains in the mitochondrial matrix. In C. europaea MCU, the N-terminal domains assemble as a dimer of dimers; in zebrafish MCU, they form an asymmetric crescent. The structures define principles that underlie ion permeation and calcium selectivity in this unusual channel.
The mitochondrial calcium uniporter is a Ca
-gated ion channel complex that controls mitochondrial Ca
entry and regulates cell metabolism. MCU and EMRE form the channel while Ca
-dependent regulation ...is conferred by MICU1 and MICU2 through an enigmatic process. We present a cryo-EM structure of an MCU-EMRE-MICU1-MICU2 holocomplex comprising MCU and EMRE subunits from the beetle Tribolium castaneum in complex with a human MICU1-MICU2 heterodimer at 3.3 Å resolution. With analogy to how neuronal channels are blocked by protein toxins, a uniporter interaction domain on MICU1 binds to a channel receptor site comprising MCU and EMRE subunits to inhibit ion flow under resting Ca
conditions. A Ca
-bound structure of MICU1-MICU2 at 3.1 Å resolution indicates how Ca
-dependent changes enable dynamic response to cytosolic Ca
signals.
The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca
channel of the inner mitochondrial ...membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 Å resolution. Using a novel assay, we demonstrate robust Ca
uptake into proteoliposomes containing the purified complex. Uptake is dependent on EMRE and also on the mitochondrial lipid cardiolipin. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.
The mitochondrial calcium uniporter is a Ca.sup.2+-gated ion channel complex that controls mitochondrial Ca.sup.2+ entry and regulates cell metabolism. MCU and EMRE form the channel while ...Ca.sup.2+-dependent regulation is conferred by MICU1 and MICU2 through an enigmatic process. We present a cryo-EM structure of an MCU-EMRE-MICU1-MICU2 holocomplex comprising MCU and EMRE subunits from the beetle Tribolium castaneum in complex with a human MICU1-MICU2 heterodimer at 3.3 Å resolution. With analogy to how neuronal channels are blocked by protein toxins, a uniporter interaction domain on MICU1 binds to a channel receptor site comprising MCU and EMRE subunits to inhibit ion flow under resting Ca.sup.2+ conditions. A Ca.sup.2+-bound structure of MICU1-MICU2 at 3.1 Å resolution indicates how Ca.sup.2+-dependent changes enable dynamic response to cytosolic Ca.sup.2+ signals.
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