scholarly journals Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

2013 ◽  
Vol 143 (1) ◽  
pp. 67-73 ◽  
Author(s):  
Ming-Feng Tsai ◽  
Dawei Jiang ◽  
Linlin Zhao ◽  
David Clapham ◽  
Christopher Miller
Keyword(s):  
Transport Mechanism ◽  
Turnover Rate ◽  
Leucine Zipper ◽  
Transmembrane Protein ◽  
Ruthenium Red ◽  
Mitochondrial Calcium Uniporter ◽  
Mitochondrial Inner Membrane ◽  
Calcium Uniporter ◽  
Ef Hand ◽  
Inner Membrane Protein

The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca2+ and K+ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca2+ fluorophore and 45Ca2+-based assays, we demonstrate directly that Letm1 is a Ca2+ transporter, with apparent affinities of cations in the sequence of Ca2+ ≈ Mn2+ > Gd3+ ≈ La3+ > Sr2+ >> Ba2+, Mg2+, K+, Na+. Kinetic analysis yields a Letm1 turnover rate of 2 Ca2+/s and a Km of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca2+/2 H+ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H+-dependent Ca2+ transport mechanism identified in intact mitochondria.

scholarly journals Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ming-Feng Tsai ◽  
Charles B Phillips ◽  
Matthew Ranaghan ◽  
Chen-Wei Tsai ◽  
Yujiao Wu ◽  
...  
Keyword(s):  
Regulatory Subunit ◽  
Mitochondrial Calcium ◽  
Transmembrane Helices ◽  
Mitochondrial Calcium Uniporter ◽  
Activating Function ◽  
Mitochondrial Inner Membrane ◽  
Calcium Uniporter ◽  
Regulatory Purpose ◽  
Inner Membrane Protein ◽  
Dual Functions

Mitochondrial Ca2+ uptake, a process crucial for bioenergetics and Ca2+ signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca2+-activated Ca2+ channel, with the Ca2+ pore formed by the MCU protein and Ca2+-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca2+ permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca2+ landscape.

scholarly journals Priority Strategy of Intracellular Ca2+ Homeostasis in Skeletal Muscle Fibers during the Multiple Stresses of Hibernation

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 42 ◽  
Author(s):  
Jie Zhang ◽  
Xiaoyu Li ◽  
Fazeela Ismail ◽  
Shenhui Xu ◽  
Zhe Wang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Leucine Zipper ◽  
Binding Capacity ◽  
Transmembrane Protein ◽  
Muscle Fibers ◽  
Vital Role ◽  
Ground Squirrels ◽  
Calcium Uniporter ◽  
Skeletal Muscle Fibers ◽  
Ef Hand

Intracellular calcium (Ca2+) homeostasis plays a vital role in the preservation of skeletal muscle. In view of the well-maintained skeletal muscle found in Daurian ground squirrels (Spermophilus dauricus) during hibernation, we hypothesized that hibernators possess unique strategies of intracellular Ca2+ homeostasis. Here, cytoplasmic, sarcoplasmic reticulum (SR), and mitochondrial Ca2+ levels, as well as the potential Ca2+ regulatory mechanisms, were investigated in skeletal muscle fibers of Daurian ground squirrels at different stages of hibernation. The results showed that cytoplasmic Ca2+ levels increased in the skeletal muscle fibers during late torpor (LT) and inter-bout arousal (IBA), and partially recovered when the animals re-entered torpor (early torpor, ET). Furthermore, compared with levels in the summer active or pre-hibernation state, the activity and protein expression levels of six major Ca2+ channels/proteins were up-regulated during hibernation, including the store-operated Ca2+ entry (SOCE), ryanodine receptor 1 (RyR1), leucine zipper-EF-hand containing transmembrane protein 1 (LETM1), SR Ca2+ ATPase 1 (SERCA1), mitochondrial calcium uniporter complex (MCU complex), and calmodulin (CALM). Among these, the increased extracellular Ca2+ influx mediated by SOCE, SR Ca2+ release mediated by RyR1, and mitochondrial Ca2+ extrusion mediated by LETM1 may be triggers for the periodic elevation in cytoplasmic Ca2+ levels observed during hibernation. Furthermore, the increased SR Ca2+ uptake through SERCA1, mitochondrial Ca2+ uptake induced by MCU, and elevated free Ca2+ binding capacity mediated by CALM may be vital strategies in hibernating ground squirrels to attenuate cytoplasmic Ca2+ levels and restore Ca2+ homeostasis during hibernation. Compared with that in LT or IBA, the decreased extracellular Ca2+ influx mediated by SOCE and elevated mitochondrial Ca2+ uptake induced by MCU may be important mechanisms for the partial cytoplasmic Ca2+ recovery in ET. Overall, under extreme conditions, hibernating ground squirrels still possess the ability to maintain intracellular Ca2+ homeostasis.

scholarly journals Different Sensitivity of Control and MICU1- and MICU2-Ablated Trypanosoma cruzi Mitochondrial Calcium Uniporter Complex to Ruthenium-Based Inhibitors

2020 ◽  
Vol 21 (23) ◽  
pp. 9316
Author(s):  
Mayara S. Bertolini ◽  
Roberto Docampo
Keyword(s):  
Trypanosoma Cruzi ◽  
Ruthenium Red ◽  
Host Cells ◽  
Mitochondrial Calcium ◽  
Low Permeability ◽  
Intact Cells ◽  
Mitochondrial Calcium Uniporter ◽  
Biochemical Characteristics ◽  
Permeabilized Cells ◽  
Calcium Uniporter

The mitochondrial Ca2+ uptake in trypanosomatids shares biochemical characteristics with that of animals. However, the composition of the mitochondrial Ca2+ uniporter complex (MCUC) in these parasites is quite peculiar, suggesting lineage-specific adaptations. In this work, we compared the inhibitory activity of ruthenium red (RuRed) and Ru360, the most commonly used MCUC inhibitors, with that of the recently described inhibitor Ru265, on Trypanosoma cruzi, the agent of Chagas disease. Ru265 was more potent than Ru360 and RuRed in inhibiting mitochondrial Ca2+ transport in permeabilized cells. When dose-response effects were investigated, an increase in sensitivity for Ru360 and Ru265 was observed in TcMICU1-KO and TcMICU2-KO cells as compared with control cells. In the presence of RuRed, a significant increase in sensitivity was observed only in TcMICU2-KO cells. However, application of Ru265 to intact cells did not affect growth and respiration of epimastigotes, mitochondrial Ca2+ uptake in Rhod-2-labeled intact cells, or attachment to host cells and infection by trypomastigotes, suggesting a low permeability for this compound in trypanosomes.

Abstract 091: Mitochondrial Calcium Currents Are Conducted By A Pore Encoded By The MCU Gene.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Dipayan Chaudhuri ◽  
Yasemin Sancak ◽  
Vamsi K Mootha ◽  
David E Clapham
Keyword(s):  
Ruthenium Red ◽  
Calcium Currents ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Atp Production ◽  
Calcium Uniporter ◽  
Selective Channel ◽  
Voltage Clamping ◽  
Open Question ◽  
Pore Domain

Mitochondrial uptake of calcium (Ca 2+ ) in cardiomyocytes is thought to regulate cell survival and match ATP production to energetic demands. Of multiple pathways allowing such entry, the mitochondrial Ca 2+ uniporter is a highly Ca 2+ -selective channel encoded by several recently-discovered genes. However, which of these several genes encodes the pore-forming subunit of the channel remains an open question. Using whole-mitoplast voltage-clamping, we show that RNAi-mediated knockdown of the mitochondrial calcium uniporter ( MCU ) gene reduces the uniporter current ( I MiCa ), whereas overexpression increases it. Mutations in the putative pore domain alter key features of I MiCa , such as its strong affinity for ruthenium red, a classic uniporter blocker. These analyses demonstrate that MCU encodes the pore-forming subunit of the uniporter channel.

scholarly journals Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) forms a Ca2+/H+ antiporter

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Juan Shao ◽  
Zhenglin Fu ◽  
Yanli Ji ◽  
Xiangchen Guan ◽  
Shang Guo ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Transmembrane Protein ◽  
Ef Hand

scholarly journals Structural characterization of the N-terminal domain of the Dictyostelium discoideum mitochondrial calcium uniporter

2019 ◽  
Author(s):  
Yuan Yuan ◽  
Chan Cao ◽  
Maorong Wen ◽  
Min Li ◽  
Ying Dong ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dictyostelium Discoideum ◽  
Self Assembly ◽  
Calcium Influx ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Terminal Domain ◽  
Calcium Uniporter

AbstractThe mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

scholarly journals Molecular basis of EMRE-dependence of the human mitochondrial calcium uniporter

2019 ◽  
Author(s):  
Melissa J.S. MacEwen ◽  
Andrew L. Markhard ◽  
Mert Bozbeyoglu ◽  
Forrest Bradford ◽  
Olga Goldberger ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Molecular Basis ◽  
Transmembrane Protein ◽  
Transmembrane Domains ◽  
Chimeric Proteins ◽  
Evolutionary Divergence ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Channel Opening ◽  
Strict Requirement

ABSTRACTThe mitochondrial uniporter is calcium-activated calcium channel complex critical for cellular signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan-specific, single-pass transmembrane protein EMRE for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of the human MCU’s dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent D. discoideum MCU (DdMCU) and H. sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in DdMCU that can be transplanted to HsMCU to render it EMRE-dependent. We call this region in human MCU the EMRE-dependence domain (EDD). Crosslinking experiments show that HsEMRE directly interacts with MCU at both of its transmembrane domains as well as the EDD. Based on previously published structures of fungal MCU homologs, the EDD segment is located distal to the calcium pore’s selectivity filter and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance

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