MICU1 occludes MCU in the mitochondrial calcium uniporter complex

Mapping Intimacies ◽

10.1101/2021.11.15.468501 ◽

2021 ◽

Author(s):

Chen-Wei Tsai ◽

Ming-Feng Tsai

Keyword(s):

Patch Clamp ◽

Xenopus Oocytes ◽

Mitochondrial Calcium ◽

Mitochondrial Matrix ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Biochemical Studies

The mitochondrial calcium uniporter imports cytoplasmic Ca2+ into the mitochondrial matrix to regulate cell bioenergetics, Ca2+ signaling, and apoptosis. The uniporter contains the pore-forming MCU subunit, an EMRE protein that binds to MCU, and the regulatory MICU1/MICU2 subunits. Structural and biochemical studies have suggested that MICU1 gates MCU by blocking and unblocking the Ca2+ pore. However, mitoplast patch-clamp experiments argue that MICU1 does not block Ca2+ transport but instead potentiates MCU. To address this direct clash of proposed MICU1 function, we applied purified MICU1 to Ca2+-conducting MCU-EMRE subcomplexes in outside-out patches excised from Xenopus oocytes. MICU1 strongly inhibits Ca2+ currents, and the inhibition is abolished by mutating an MCU-interacting K126 residue in MICU1. Further experiments show that MICU1 block was not observed in mitoplasts because MICU1 dissociates from the uniporter complex. These results firmly establish that MICU1 shuts the uniporter in resting cellular conditions.

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Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes

The Journal of General Physiology ◽

10.1085/jgp.201812015 ◽

2018 ◽

Vol 150 (7) ◽

pp. 1035-1043 ◽

Cited By ~ 4

Author(s):

Chen-Wei Tsai ◽

Ming-Feng Tsai

Keyword(s):

Xenopus Oocytes ◽

Single Channel ◽

Mitochondrial Calcium ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Electrophysiological Recordings ◽

Electrode Voltage ◽

Genes Encoding ◽

Functional Mechanisms ◽

Inwardly Rectifying

The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+. Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.

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Patch-Clamp Analysis of the Mitochondrial Calcium Uniporter

Calcium Signalling - Methods in Molecular Biology ◽

10.1007/978-1-4939-9018-4_7 ◽

2019 ◽

pp. 75-86 ◽

Cited By ~ 5

Author(s):

Vivek Garg ◽

Yuriy Y. Kirichok

Keyword(s):

Patch Clamp ◽

Mitochondrial Calcium ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter

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Mitochondrial Calcium Entry Is Essential in Agonist-Induced Procoagulant Platelet Formation

Blood ◽

10.1182/blood.v128.22.3721.3721 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3721-3721

Author(s):

Andaleb Kholmukhamedov ◽

Rae Janecke ◽

Hyojung Choo ◽

Shawn M Jobe

Keyword(s):

Mitochondrial Membrane ◽

Transmembrane Potential ◽

Calcium Entry ◽

Mitochondrial Transmembrane Potential ◽

Mitochondrial Calcium ◽

Mitochondrial Matrix ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Platelet Formation

Abstract Procoagulant platelets are a subpopulation of activated platelets with high-level phosphatidylserine (PSer) externalization. When initiated by co-stimulation with thrombin (THR) and a GPVI agonist, such as convulxin (CVX) or collagen, both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant platelet phenotype. The intracellular mechanisms coordinating these processes remain unclear. Here we investigated the role of the mitochondrial calcium uniporter in the regulation of procoagulant platelet formation. The flux of calcium from the cytosol to the mitochondrial matrix is limited by the ion-impermeable inner mitochondrial membrane. Initially, a pharmacologic approach was utilized to investigate the importance of mitochondrial calcium entry and elevation. Washed human or murine platelets were stimulated with THR and/or CVX in physiologic buffer with 2 mM CaCl2. Mitochondrial transmembrane potential was evaluated using 0.5 μM tetramethylrhodamine methylester (TMRM). Limitation of mitochondrial calcium entry either through alteration of pre-stimulatory mitochondrial membrane potential (i.e. inhibition of F0F1 ATP-ase, mitochondrial uncoupling etc.) or using the ruthenium red analog (Ru360) abrogated both mPTP and procoagulant platelet formation consistent with the hypothesis that elevated calcium levels within the mitochondrial matrix drives these processes. The molecular pathway mediating this rapid mechanism of calcium entry was investigated using mitochondrial calcium uniporter (MCU) null platelets. MCU is a transmembrane ion channel that allows the passage of Ca2+ from the cytosol into the mitochondrial matrix. Neither platelet aggregatory ability nor granule release was altered in response to single or dual-agonist stimulation. In contrast, procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation as measured by loss of mitochondrial transmembrane potential. Whereas the number of annexin V positive platelets decreased from 60% in WT to 32% in MCU null (p<0.05, n=4). Thus, these results identify a key role of mitochondrial calcium uptake channel in the regulation of strong agonist-initiated procoagulant platelet formation and suggest a novel pharmacologic target that could be used the treatment of procoagulant-platelet related pathologies. Disclosures No relevant conflicts of interest to declare.

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