G6PC3 Deficiency Associated with Congenital Neutropenia and Enterocolitis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2170-2170
Author(s):  
Jana Diestelhorst ◽  
Daniel Kotlarz ◽  
Giridharan Appaswamy ◽  
Rita Beier ◽  
Peter M Krawitz ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Sequence Variant ◽  
Neutrophil Granulocytes ◽  
Inner Mitochondrial Membrane ◽  
Congenital Neutropenia ◽  
Exon 2 ◽  
Healthy Sibling ◽  
Tract Infections

Abstract Abstract 2170 Glucose-6-phosphatase catalytic subunit 3 (G6PC3) deficiency causes congenital neutropenia in conjunction with various cardiac or urogenital developmental aberrations. We here describe a consanguineous pedigree of maroccan descent. Four patients presented with early-onset inflammatory bowel diseases associated with severe congenital neutropenia. Two children died in early childhood, two patients (19-years old female, 17-years old male) are alive with signs of recurrent infections (mouth ulcera, otitis, upper and lower respiratory tract infections) and refractory Crohn's like enterocolitis. Mutations in the Glucose-6-phosphate translocase (SLC37A4) gene were excluded. In an attempt to discover the underlying molecular pathophysiology, we performed SNP-based homozygosity mapping followed by in depth next-generation exome sequencing. We identified two genes with potentially deleterious homozygous sequence variations: P2RY11 (exon 13; c.C2160G, p.Y720X), encoding a protein with antiapoptotic function, and G6PC3 (exon 2; c.C323T, p.P108L). The G6PC3 mutation segregates with the disease phenotype, whereas the P2RY11 variant was also found in a healthy sibling. Functional experiments in patient's neutrophil granulocytes showed an accelerated oxidative burst capacity (OxyBurst Assay, Phagoburst Assay). Furthermore, G6PC3-deficient individuals showed a rapid dissipation of the mitochondrial membrane potential upon treatment with valinomycin, whereas the inner mitochondrial membrane potential in neutrophils from healthy individuals was maintained. We observed increased apoptosis in G6PC3-deficient neutrophils upon exposure to staurosporine. Interestingly, increased apoptosis was also seen in granulocytes from the healthy sibling with the P2RY11 sequence variant, suggesting that P2RY11 deficiency may aggravate the phenotype of G6PC3 deficiency. In summary, our study indicates that the clinical spectrum of G6PC3 deficiency may be more diverse than previously appreciated and that P2RY11 may have a modifying effect on G6PC3-deficient neutrophil granulocytes. J.D. and D. K. contributed equally to this work and should be considered aequo loco. Disclosures: Schäffer: National Institues of Health: Employment; Intramural Research Program of the National Institues of Health, NLM: Research Funding.

atpif1 regulates Mitochondrial Heme Synthesis In Developing Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 163-163
Author(s):  
Dhvanit I Shah ◽  
Naoko Takahasi-Makise ◽  
Iman Schultz ◽  
Eric L Pierce ◽  
Liangtao Li ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Positional Cloning ◽  
Conflicts Of Interest ◽  
Microcytic Anemia ◽  
Mitochondrial Atpase ◽  
Cluster Assembly ◽  
Inner Mitochondrial Membrane ◽  
Heme Synthesis

Abstract Abstract 163 Iron plays a key role as a cofactor in many fundamental metabolic processes, which require heme synthesis and Fe/S cluster assembly in the mitochondria. Defects in the transport of iron into the mitochondria would lead to anemias due to a deficiency in heme and hemoglobin synthesis. Here we describe a zebrafish genetic mutant, pinotage (pnttq209), which exhibits a profound hypochromic, microcytic anemia. Erythrocytes from pnt mutants have a defect in hemoglobinization and decreased red cell indices (mean corpuscular volume and hemoglobin content, hematocrit, hemoglobin concentration). Through positional cloning, we showed that the mitochondrial ATPase Inhibitory Factor 1 (atpif1), which regulates the inner mitochondrial membrane potential, is the gene disrupted in pnt. The identity of the pnt gene was verified by: (a) decreased atpif1 steady-state mRNA in pnt mutants, (b) phenocopying the anemia with anti-sense atpif1 morpholinos, (c) functional complementation of the anemia with atpif1 cRNA, and (d) a genetic polymorphism in the 3'UTR co-segregating with the mutant phenotype that destabilizes the atpif1 mRNA. Consistent with the conserved function of atpif1 in higher vertebrates, the silencing of the murine ortholog of atpif1 in Friend mouse erythroleukemia (MEL) cells showed a defect in hemoglobinization by o-dianisidine staining and reduction of 59Fe incorporation into heme in 59Fe-metabolically labeled cells. Moreover, Atpif1 knockdown destabilizes their mitochondrial membrane potential and volume. Therefore, the identification of atpif1 in pnt functionally demonstrates the role of atpif1 in regulating the proton motive gradient across the inner mitochondrial membrane for mitochondrial iron incorporation in heme biosynthesis. These results uncover a novel hematopoiesis-related function of atpif1, which will directly contribute to our understanding and potential treatment of human congenital and acquired anemias. Disclosures: No relevant conflicts of interest to declare.

107 DIFFERENCES IN THE INNER MITOCHONDRIAL MEMBRANE POTENTIAL BETWEEN NON-CULTURED AND CULTURED PORCINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 159
Author(s):  
M. Romek ◽  
B. Gajda ◽  
M. Rolka ◽  
Z. Smorag
Keyword(s):  
Fatty Acids ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Developmental Competence ◽  
Critical Event ◽  
Inner Mitochondrial Membrane ◽  
Oxidation Rates

In comparison to in vivo derived pig embryos, in vitro culture conditions produce embryos with altered metabolic rates of carbohydrates and fatty acids (Romek M et al. 2010 Theriogenology 74, 265–276), which may compromise embryo viability. Because various energy substrates are metabolized via several aerobic pathways leading to generation of the inner mitochondrial membrane potential (ΔΨm), value of ΔΨm is a key indicator of embryo metabolic activity, closely related to oxygen consumption and cellular energy needs. Therefore, the aim of this study was to compare ΔΨm between non-cultured and cultured pig embryos during early development. The non-cultured embryos were obtained from 6-month-old gilts, whereas those derived in vitro were cultured from zygotes to the appropriate stage in North Carolina State University 23 (NCSU-23) medium supplemented with 4 mg mL–1 of bovine serum albumin. The ΔΨm measurements were carried out on both non-cultured and cultured 4 to 8 cell embryos, morulae, blastocysts and late blastocysts. For this, embryos were labelled with 0.5 μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and then with 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 10°C. Using a LSM 510 Meta Zeiss confocal microscope, we measured the amounts of fluorescence (IMtOR and IMtDR) emitted from embryos and values of ΔΨm were estimated as the IMtOR/IMtDR ratios. The results were analysed by ANOVA and Tukey's test. From the zygote to morula stages, ΔΨm remained unchanged and did not differ between developmentally matched non-cultured and cultured embryos (P < 0.001). The value of ΔΨm increased significantly (P < 0.05) from 0.90 ± 0.26 arbitrary units (a.u.) for morulae to 3.92 ± 0.63 and 2.06 ± 0.38 a.u. for non-cultured and cultured early blastocysts, respectively. Whereas the mean value of ΔΨm was almost 2 times higher in non-cultured than in cultured early blastocysts, the mitochondrial membrane potential was statistically similar (P < 0.05) in the in vivo derived (2.10 ± 0.37 a.u.) compared to cultured (1.87 ± 0.30 a.u.) blastocysts. The lower ΔΨm in cultured early blastocysts may be explained by several-fold higher glucose concentration in NCSU-23 medium than in the oviductal fluid. It was reported that high levels of glucose decreases the Krebs cycle metabolism of pyruvate, glutamine, and glucose, and reduces oxidation rates of fatty acids in cultured pig embryos in comparison with in vivo counterparts. Hence, this impaired metabolism reflected by decreased ΔΨm may be responsible for insufficient energy production and reduced developmental competence of cultured early blastocysts. Therefore, because embryo-cavitation is a critical event in pig development, further effort should be focused on proper blastocyst culture. Research was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.

A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria

2016 ◽  
Vol 473 (23) ◽  
pp. 4457-4471 ◽  
Author(s):  
Anna Kicinska ◽  
Bartlomiej Augustynek ◽  
Bogusz Kulawiak ◽  
Wieslawa Jarmuszkiewicz ◽  
Adam Szewczyk ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Potassium Channel ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Dermal Fibroblast ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
K Channel ◽  
Inner Mitochondrial Membrane ◽  
Bkca Channels

Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca2+-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca2+ and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and β-subunits (predominantly the β3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca2+-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts.

scholarly journals Adenine Nucleotide Translocase 1 Expression Is Coupled to the HSP27-Mediated TLR4 Signaling in Cardiomyocytes

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1588 ◽  
Author(s):  
Julia Winter ◽  
Elke Hammer ◽  
Jacqueline Heger ◽  
Heinz-Peter Schultheiss ◽  
Ursula Rauch ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Adenine Nucleotide ◽  
Heart Tissue ◽  
Neonatal Rat ◽  
Adenine Nucleotide Translocase ◽  
Inner Mitochondrial Membrane ◽  
Tlr4 Signaling ◽  
Rat Cardiomyocytes

The cardiac-specific overexpression of the adenine nucleotide translocase 1 (ANT1) has cardioprotective effects in various experimental heart disease models. Here, we analyzed the link between ANT1 expression and heat shock protein 27 (HSP27)-mediated toll-like receptor 4 (TLR4) signaling, which represents a novel communication pathway between mitochondria and the extracellular environment. The interaction between ANT1 and HSP27 was identified by co-immunoprecipitation from neonatal rat cardiomyocytes. ANT1 transgenic (ANT1-TG) cardiomyocytes demonstrated elevated HSP27 expression levels. Increased levels of HSP27 were released from the ANT1-TG cardiomyocytes under both normoxic and hypoxic conditions. Extracellular HSP27 stimulated TLR4 signaling via protein kinase B (AKT). The HSP27-mediated activation of the TLR4 pathway was more pronounced in ANT1-TG cardiomyocytes than in wild-type (WT) cardiomyocytes. HSP27-specific antibodies inhibited TLR4 activation and the expression of HSP27. Inhibition of the HSP27-mediated TLR4 signaling pathway with the TLR4 inhibitor oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) reduced the mitochondrial membrane potential (∆ψm) and increased caspase 3/7 activity, which are both markers for cell stress. Conversely, treating cardiomyocytes with recombinant HSP27 protein stimulated TLR4 signaling, induced HSP27 and ANT1 expression, and stabilized the mitochondrial membrane potential. The activation of HSP27 signaling was verified in ischemic ANT1-TG heart tissue, where it correlated with ANT1 expression and the tightness of the inner mitochondrial membrane. Our study shows a new mechanism by which ANT1 is part of the cardioprotective HSP27-mediated TLR4 signaling.

scholarly journals Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function

1998 ◽  
Vol 275 (5) ◽  
pp. H1567-H1576 ◽  
Author(s):  
Ekshon L. Holmuhamedov ◽  
Sofija Jovanović ◽  
Petras P. Dzeja ◽  
Aleksandar Jovanović ◽  
Andre Terzic
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Function ◽  
Mitochondrial Membrane ◽  
Membrane Depolarization ◽  
Cardiac Mitochondria ◽  
Inner Mitochondrial Membrane ◽  
Induced Membrane ◽  
Channel Opener ◽  
Matrix Volume

Discovered in the cardiac sarcolemma, ATP-sensitive K+(KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATPchannel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of −180 ± 15 mV, K+ channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome cand adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.

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