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1.
Proc Natl Acad Sci U S A ; 118(27)2021 07 06.
Article in English | MEDLINE | ID: mdl-34215698

ABSTRACT

Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de-N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2-like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum-associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCFFBS2 in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of "glycan-less" NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCFFBS2 accounts for the pathogenesis resulting from NGLY1 deficiency.


Subject(s)
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Proteasome Endopeptidase Complex/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Sugars/metabolism , Animals , Behavior, Animal , Cell Death , Cell Nucleus/metabolism , Cell Proliferation , Cytosol/metabolism , HCT116 Cells , HeLa Cells , Humans , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Motor Activity , Mutation/genetics , Nuclear Respiratory Factor 1/metabolism , Polysaccharides/metabolism , Protein Transport , Ubiquitination
2.
J Cell Biol ; 219(9)2020 09 07.
Article in English | MEDLINE | ID: mdl-32556086

ABSTRACT

Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson's disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN-ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.


Subject(s)
Autophagy-Related Proteins/metabolism , Cell Cycle Proteins/metabolism , Membrane Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondria/physiology , Mitophagy/physiology , Ubiquitination/physiology , Vesicular Transport Proteins/metabolism , Animals , Autophagy/physiology , Carrier Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , HCT116 Cells , HEK293 Cells , HeLa Cells , Humans , Mammals/metabolism , Mammals/physiology , Protein Kinases/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism
3.
Proc Natl Acad Sci U S A ; 114(32): 8574-8579, 2017 08 08.
Article in English | MEDLINE | ID: mdl-28743755

ABSTRACT

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.


Subject(s)
Autophagy/physiology , Glycoproteins/metabolism , Lysosomes/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Ubiquitination/physiology , Glycoproteins/genetics , HeLa Cells , Humans , Lysosomes/genetics , SKP Cullin F-Box Protein Ligases/genetics
4.
Pflugers Arch ; 468(5): 837-47, 2016 05.
Article in English | MEDLINE | ID: mdl-26843093

ABSTRACT

Acidification of the resorption pits, which is essential for dissolving bone, is produced by secretion of protons through vacuolar H(+)-ATPases in the plasma membrane of bone-resorbing cells, osteoclasts. Consequently, osteoclasts face highly acidic extracellular environments, where the pH gradient across the plasma membrane could generate a force driving protons into the cells. Proton influx mechanisms during the acid exposure are largely unknown, however. In this study, we investigated extracellular-acid-inducible proton influx currents in osteoclast-like cells derived from a macrophage cell line (RAW264). Decreasing extracellular pH to <5.5 induced non-ohmic inward currents. The reversal potentials depended on the pH gradients across the membrane and were independent of concentrations of Na(+), Cl(-), and HCO3 (-), suggesting that they were carried largely by protons. The acid-inducible proton influx currents were not inhibited by amiloride, a widely used blocker for cation channels/transporters, or by 4,4'-diisothiocyanato-2,2'-stilbenesulfonate(DIDS) which blocks anion channels/transporters. Additionally, the currents were not significantly affected by V-ATPase inhibitors, bafilomycin A1 and N,N'-dicyclohexylcarbodiimide. Extracellular Ca(2+) (10 mM) did not affect the currents, but 1 mM ZnCl2 decreased the currents partially. The intracellular pH in the vicinity of the plasma membrane was dropped by the acid-inducible H(+) influx currents, which caused overshoot of the voltage-gated H(+) channels after removal of acids. The H(+) influx currents were smaller in undifferentiated, mononuclear RAW cells and were negligible in COS7 cells. These data suggest that the acid-inducible H(+) influx (H(+) leak) pathway may be an additional mechanism modifying the pH environments of osteoclasts upon exposure to strong acids.


Subject(s)
Action Potentials , Ion Channels/metabolism , Osteoclasts/metabolism , Protons , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , COS Cells , Calcium/pharmacology , Cell Membrane/drug effects , Cell Membrane/metabolism , Chlorides/pharmacology , Chlorocebus aethiops , Hydrogen-Ion Concentration , Ion Channels/drug effects , Mice , Zinc Compounds/pharmacology
5.
Eukaryot Cell ; 14(10): 976-82, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26150415

ABSTRACT

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.


Subject(s)
Amino Acid Motifs/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Endosomal Sorting Complexes Required for Transport/ultrastructure , Protein Binding , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/ultrastructure
6.
Proc Natl Acad Sci U S A ; 112(15): 4630-5, 2015 Apr 14.
Article in English | MEDLINE | ID: mdl-25827227

ABSTRACT

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.


Subject(s)
Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitinated Proteins/metabolism , Ubiquitination , Amino Acid Sequence , Antibodies/immunology , Base Sequence , F-Box Proteins/genetics , F-Box Proteins/immunology , F-Box Proteins/metabolism , Glycine/genetics , Glycine/metabolism , HEK293 Cells , Humans , Immunoblotting/methods , Immunoprecipitation/methods , Lysine/genetics , Lysine/metabolism , Molecular Sequence Data , Oligopeptides/genetics , Oligopeptides/immunology , Oligopeptides/metabolism , Protein Binding/immunology , Proteome/genetics , Proteome/immunology , Proteome/metabolism , Proteomics , Reproducibility of Results , Substrate Specificity , Tandem Mass Spectrometry/methods , Trypsin/genetics , Trypsin/metabolism , Ubiquitin/genetics , Ubiquitin/immunology , Ubiquitin-Protein Ligases/genetics , Ubiquitinated Proteins/genetics
7.
FEBS Lett ; 589(5): 576-80, 2015 Feb 27.
Article in English | MEDLINE | ID: mdl-25625920

ABSTRACT

We identified a yeast mutant with temperature-sensitive growth defects that were rescued by VCP expression. The mutation occurred in GPI10, which encodes a mannosyl transferase for glycosylphosphatidylinositol anchor formation in the endoplasmic reticulum, and caused a Gly469Glu substitution in Gpi10. The mutant exhibited increased unfolded protein response, which was partially rescued by VCP or Cdc48, and showed sensitivity against cell-wall stressors, which were not rescued by VCP. These results suggest a potential link between VCP/Cdc48 and Gpi10 functions in the control of cell growth.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Mannosyltransferases/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/genetics , Cell Cycle Proteins/genetics , Endoplasmic Reticulum/metabolism , Mannosyltransferases/genetics , Membrane Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Temperature , Valosin Containing Protein
8.
J Biol Chem ; 289(31): 21760-9, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24962567

ABSTRACT

Yeast Rfu1 (regulator for free ubiquitin chain 1) localizes to endosomes and plays a role in ubiquitin homeostasis by inhibiting the activity of Doa4. We show that Bro1, a member of the class E vacuolar protein sorting proteins that recruits Doa4 to endosomes and stimulates Doa4 deubiquitinating activity, also recruits Rfu1 to endosomes for involvement in ubiquitin homeostasis. This recruitment was mediated by the direct interaction between a region containing the YPEL motif in Rfu1 and the V domain in Bro1, which could be analogous to the interaction between the mammalian Alix V domain and YPXnL motifs of viral and cellular proteins. Furthermore, overexpression of Bro1, particularly the V domain, prevented Rfu1 degradation in response to heat shock. Thus, Bro1, a Doa4 positive regulator, regulated Rfu1, a negative regulator of Doa4. Rfu1 degradation partly involved the proteasome and a ubiquitin ligase Rsp5, suggesting that Rfu1 stability was regulated by ubiquitin-proteasome pathways.


Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Homeostasis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology , Ubiquitin/metabolism , Endosomal Sorting Complexes Required for Transport/physiology , Proteolysis
9.
Genes Cells ; 18(12): 1131-43, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24215292

ABSTRACT

VCP/p97 is a hexameric ring-shaped AAA(+) ATPase that participates in various ubiquitin-associated cellular functions. Mis-sense mutations in VCP gene are associated with the pathogenesis of two inherited diseases: inclusion body myopathy associated with Paget's disease of the bone and front-temporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). These pathogenic VCPs have higher affinities for several cofactors, including Npl4, Ufd1 and p47. In Parkin-dependent mitochondrial quality control systems, VCP migrates to damaged mitochondria (e.g., those treated with uncouplers) to aid in the degradation of mitochondrial outer membrane proteins and to eliminate mitochondria. We showed that endogenous Npl4 and p47 also migrate to mitochondria after uncoupler treatment, and Npl4, Ufd1 or p47 silencing causes defective mitochondria clearance after uncoupler treatment. Moreover, pathogenic VCPs show impaired migration to mitochondria, and the exogenous pathogenic VCP expression partially inhibits Npl4 and p47 localization to mitochondria. These results suggest that the increased affinities of pathogenic VCPs for these cofactors cause the impaired movement of pathogenic VCPs. In adult flies, exogenous expression of wild-type VCP, but not pathogenic VCPs, reduces the number of abnormal mitochondria in muscles. Failure of pathogenic VCPs to function on damaged mitochondria may be related to the pathogenesis of IBMPFD and ALS.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Mitochondria/metabolism , Nuclear Proteins/metabolism , Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Vesicular Transport , Adenosine Triphosphatases/genetics , Amyotrophic Lateral Sclerosis/genetics , Animals , Animals, Genetically Modified , Cell Cycle Proteins/genetics , Drosophila , Frontotemporal Dementia/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Mitochondria/pathology , Muscular Dystrophies, Limb-Girdle/genetics , Mutation, Missense , Myositis, Inclusion Body/genetics , Osteitis Deformans/genetics , Valosin Containing Protein
10.
J Physiol ; 591(23): 5851-66, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24081153

ABSTRACT

Voltage-gated proton channels (H(+) channels) are highly proton-selective transmembrane pathways. Although the primary determinants for activation are the pH and voltage gradients across the membrane, the current amplitudes fluctuate often when these gradients are constant. The aim of this study was to investigate the role of the intracellular pH (pHi) in regulating the availability of H(+) channels in osteoclasts and microglia. In whole-cell clamp recordings, the pHi was elevated after exposure to NH4Cl and returned to the control level after washout. However, the H(+) channel conductance did not recover fully when the exposure was prolonged (>5 min). Similar results were observed in osteoclasts and microglia, but not in COS7 cells expressing a murine H(+) channel gene (mVSOP). As other electrophysiological properties, like the gating kinetics and voltage dependence for activation, were unchanged, the decreases in the H(+) channel conductance were probably due to the decreases in H(+) channels available at the plasma membrane. The decreases in the H(+) channel conductances were accompanied by reductions in the cell capacitance. Exposure to NH4Cl increased the uptake of the endocytosis marker FM1-43, substantiating the idea that pHi increases facilitated endocytosis. In osteoclasts, whose plasma membrane expresses V-ATPases and H(+) channels, pHi increases by these H(+)-transferring molecules in part facilitated endocytosis. The endocytosis and decreases in the H(+) channel conductance were reduced by dynasore, a dynamin blocker. These results suggest that pHi increases in osteoclasts and microglia decrease the numbers of H(+) channels available at the plasma membrane through facilitation of dynamin-dependent endocytosis.


Subject(s)
Cell Membrane/physiology , Endocytosis/physiology , Ion Channels/physiology , Microglia/physiology , Osteoclasts/physiology , Ammonium Chloride/pharmacology , Animals , Benzopyrans/pharmacology , COS Cells , Cell Line , Dynamins/antagonists & inhibitors , Dynamins/physiology , Fluorescent Dyes/pharmacology , Hydrazones/pharmacology , Hydrogen-Ion Concentration , Mice , Microglia/chemistry , Microglia/drug effects , Naphthols/pharmacology , Osteoclasts/chemistry , Osteoclasts/drug effects , Pyridinium Compounds/pharmacology , Quaternary Ammonium Compounds/pharmacology , Rats , Rhodamines/pharmacology
11.
J Struct Biol ; 179(2): 93-103, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22728077

ABSTRACT

VCP/p97/Cdc48 is a hexameric ring-shaped AAA ATPase that participates in a wide variety of cellular functions. VCP is a very abundant protein in essentially all types of cells and is highly conserved among eukaryotes. To date, 19 different single amino acid-substitutions in VCP have been reported to cause IBMPFD (inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia), an autosomal dominant inherited human disease. Moreover, several similar single amino acid substitutions have been proposed to associate with a rare subclass of familial ALS. The mechanisms by which these mutations contribute to the pathogenesis are unclear. To elucidate potential functional differences between wild-type and pathogenic VCPs, we expressed both VCPs in yeast cdc48 mutants. We observed that all tested pathogenic VCPs suppressed the temperature-sensitive phenotype of cdc48 mutants more efficiently than wild-type VCP. In addition, pathogenic VCPs, but not wild-type VCP, were able to rescue a lethal cdc48 disruption. In yeast, pathogenic VCPs, but not wild-type VCP, formed apparent cytoplasmic foci, and these foci were transported to budding sites by the Myo2/actin-mediated transport machinery. The foci formation of pathogenic VCPs appeared to be associated with their suppression of the temperature-sensitive phenotype of cdc48 mutants. These results support the idea that the pathogenic VCP mutations create dominant gain-of-functions rather than a simple loss of functional VCP. Their unique properties in yeast could provide a convenient drug-screening system for the treatment of these diseases.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Frontotemporal Dementia/enzymology , Muscular Dystrophies, Limb-Girdle/enzymology , Myositis, Inclusion Body/enzymology , Osteitis Deformans/enzymology , Yeasts/enzymology , Yeasts/growth & development , Adenosine Triphosphatases/genetics , Cell Cycle Proteins/genetics , Flow Cytometry , Genetic Complementation Test , Humans , Mutation , Valosin Containing Protein , Yeasts/genetics
12.
J Physiol ; 590(4): 827-44, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22183729

ABSTRACT

Voltage-gated proton channels play crucial roles during the respiratory burst in phagocytes, such as microglia. As local anaesthetics have a variety of anti-inflammatory properties, including inhibition of phagocytosis, they may act on the proton channels. Most local anaesthetics are tertiary amines and may affect proton channels through modification of pH(i) as weak bases. To test these hypotheses, the effects of lidocaine and bupivacaine on proton channels were examined in a rat microglial cell line (GMI-R1) as a function of pH(o) and pH(i). Both lidocaine and bupivacaine reversibly decreased the current, with IC(50) values of ∼1.2 and ∼0.5 mM, respectively, at pH(o)/pH(i) 7.3/5.5. The inhibition was enhanced with either pH(o) increase or pH(i) decrease, suggesting that the protonation of the base forms inside the cell contributed to the inhibitory effects. Both local anaesthetics shifted the reversal potentials to more positive voltages, indicating increases in pH(i). The potencies of inhibition were correlated well with the degree of increase in pH(i). The lidocaine-induced inhibition was eliminated when the pH(i) increases were cancelled by co-application of a weak acid, butyrate. The cytosolic alkalizations by lidocaine and bupivacaine were confirmed using a pH-sensitive fluorescent dye, BCECF, in non-voltage-clamped cells. Furthermore, chemiluminescence measurement proved that both anaesthetics inhibited production of reactive oxygen species by the cells. In conclusion, lidocaine and bupivacaine inhibit proton channels primarily by the weak base mechanism via an increase in pH(i). This is a novel mechanism underlying actions of local anaesthtics.


Subject(s)
Anesthetics, Local/pharmacology , Bupivacaine/pharmacology , Ion Channels/drug effects , Lidocaine/pharmacology , Protons , Animals , Cell Line , Hydrogen-Ion Concentration , Ion Channels/physiology , Microglia/drug effects , Microglia/physiology , Rats , Reactive Oxygen Species/metabolism
13.
Am J Physiol Cell Physiol ; 299(3): C570-8, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20592242

ABSTRACT

In osteoclasts, elevation of extracellular Ca2+ is an endogenous signal that inhibits bone resorption. We recently found that an elevation of extracellular Ca2+ decreased proton extrusion through the plasma membrane vacuolar H+-ATPase (V-ATPase) rapidly. In this study we investigated mechanisms underlying this early Ca2+-sensing response, particularly in reference to the activity of the plasma membrane V-ATPase and to membrane retrieval. Whole cell clamp recordings allowed us to measure the V-ATPase currents and the cell capacitance (C(m)) simultaneously. C(m) is a measure of cell surface. Extracellular Ca2+ (2.5-40 mM) decreased C(m) and the V-ATPase current simultaneously. The decreased C(m), together with the enhanced uptake of a lipophilic dye (FM1-43), indicated that Ca2+ facilitated endocytosis. The endocytosis was blocked by dynamin inhibitors (dynasore and dynamin-inhibitory peptide), by small interfering RNA (siRNA) targeting for dynanmin-2 and also by bafilomycin A(1), a blocker of V-ATPases. The extracellular Ca2+-induced endocytosis and inhibition of the V-ATPase current were diminished by a phospholipase C inhibitor (U73122) and siRNA targeting for phospholipase C gamma2 subunit. Holding the cytosolic Ca2+ at either high (0.5-5 microM) or low levels or inhibiting calmodulin by an inhibitor (W7) or an antibody (anti-CaM) decreased the stimulated endocytosis and the inhibition of the V-ATPase current. These data suggest that extracellular Ca2+ facilitated dynamin- and V-ATPase-dependent endocytosis in association with an inhibition of the plasma membrane V-ATPase. Phospholipase C, cytosolic Ca2+, and calmodulin were involved in the signaling pathways. Membrane retrieval and the plasma membrane V-ATPase activity may cooperate during the early phase of Ca2+-sensing response in osteoclasts.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , Endocytosis , Osteoclasts/metabolism , Type C Phospholipases/physiology , Vacuolar Proton-Translocating ATPases/antagonists & inhibitors , Animals , Calmodulin/metabolism , Cell Line , Cytosol/metabolism , Dynamins/metabolism , Electric Capacitance , Extracellular Space/metabolism , Mice , Patch-Clamp Techniques , Signal Transduction , Vacuolar Proton-Translocating ATPases/physiology
14.
Pflugers Arch ; 455(5): 829-38, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17876602

ABSTRACT

Voltage-gated proton (H+) channels play a pivotal role in compensating charge and pH imbalances during respiratory bursts in phagocytes. Lactic acidosis is a clinically important metabolic condition accompanying various tissue disorders in which the extracellular pH and the intracellular pH often change in parallel. In this study, we investigated the responses of the H+ channel in microglia to lactate-induced pH disturbances using the perforated-patch recordings. Na-lactate (pH 6.8) acidified the cells and activated the H+ channel within 5 min. This early activation was correlated with increases in the pH gradient across the plasma membrane (DeltapH) and was dose-dependent over a concentration range of 10-150 mM. At 10 mM, the change in DeltapH was only slight, but the H+ currents continued to increase over an hour after the cell acidosis was stabilized. Prolonged exposure to lactate (10-20 mM, >1 h) increased the amplitude by two to threefold. The late activation was not explained by increased DeltapH but by changes in the property of the channel per se. Pretreatment with staurosporine and chelerythrine, inhibitors for protein kinase C, prevented the late activation. These results suggest that the H+ channel could be activated greatly during long-lasting lactic acidosis through both DeltapH-dependent and -independent mechanisms.


Subject(s)
Acidosis, Lactic/physiopathology , Hydrogen-Ion Concentration , Ion Channel Gating/physiology , Ion Channels/physiology , Microglia/physiology , Protons , Acid-Base Equilibrium/physiology , Animals , Cell Line , Cell Size , Electric Stimulation , Enzyme Inhibitors/pharmacology , Lactic Acid/pharmacology , Microglia/cytology , Patch-Clamp Techniques , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Staurosporine/pharmacology
15.
J Physiol ; 576(Pt 2): 417-25, 2006 Oct 15.
Article in English | MEDLINE | ID: mdl-16901940

ABSTRACT

The vacuolar-type H(+)-ATPase (V-ATPase) in the plasma membrane of a variety of cells serves as an acid-secreting pathway, and its activity is closely related to cellular functions. Massive proton secretion often leads to electrolyte disturbances in the vicinity of the cell and may in turn affect the activity of the V-ATPase. We characterized, for the first time, the proton currents mediated by plasmalemmal V-ATPase in murine osteoclast-like cells and investigated its activity over a wide range of pH gradients across the membrane (DeltapH = extracellular pH - intracellular pH). The V-ATPase currents were identified as outward H(+) currents and were dependent on ATP and sensitive to the inhibitors bafilomycin A(1) and N,N'-dicyclohexylcarbodiimide. Although H(+) was transported uphill, the electrochemical gradient for H(+) affected the current. The currents were increased by elevating DeltapH and depolarization, and were reduced by lowering DeltapH and hyperpolarization. Elevation of extracellular Ca(2+) (5-40 mm) diminished the currents in a dose-dependent manner and made the voltage dependence more marked. Extracellular Mg(2+) mimicked the inhibition. With 40 mm Ca(2+), the currents decreased to < 40% at 0 mV and to < 10% at about -80 mV. Increases in the intracellular Ca(2+) (0.5-5 microm) did not affect the current. The data suggest that acid secretion through the plasmalemmal V-ATPase is regulated by a combination of the pH gradient, the membrane potential and the extracellular divalent cations. In osteoclasts, the activity-dependent accumulation of acids and Ca(2+) in the closed extracellular compartment might serve as negative feedback signals for regulating the V-ATPase.


Subject(s)
Calcium/pharmacology , Cell Membrane/enzymology , Proton-Translocating ATPases/physiology , Protons , Animals , Cell Line , Dicyclohexylcarbodiimide/pharmacology , Dose-Response Relationship, Drug , Electrophysiology , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Intracellular Membranes/enzymology , Macrolides/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Osteoclasts/drug effects , Osteoclasts/enzymology , Osteoclasts/physiology , Patch-Clamp Techniques
16.
J Bone Miner Res ; 18(11): 2069-76, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14606521

ABSTRACT

UNLABELLED: The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. INTRODUCTION: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. METHODS: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). RESULTS AND CONCLUSIONS: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (delta(pH)), indicating that the H+ channel changed its activity by sensing delta(pH). The reversal potential (V(rev)) was a valuable tool for the real-time monitoring of delta(pH) in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 microM) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of V(rev), suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K(+)-rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In approximately 80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the delta(pH) changes, implying that both delta(pH)-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward V(rev). These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C.


Subject(s)
Acidosis/chemically induced , Ion Channel Gating/drug effects , Ion Channels/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Acidosis/metabolism , Animals , Electric Conductivity , Hydrogen-Ion Concentration , Male , Membrane Potentials/drug effects , Mice , Patch-Clamp Techniques , Protons
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