Publications by authors named "Miyuki Nishi"

54 Publications

Enhanced Ca handling in thioglycolate-elicited peritoneal macrophages.

Cell Calcium 2021 Feb 21;96:102381. Epub 2021 Feb 21.

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 606-8501, Japan. Electronic address:

In macrophage biology, resident peritoneal macrophages (RPMs) and thioglycolate-elicited peritoneal macrophages (TGPMs) have been traditionally utilized as primary cultured models. RPMs and TGPMs exhibit distinct morphological, functional and metabolic characteristics, although it remains unclear how cellular Ca handling differs between them. In our Fura-2 Ca imaging, TGPMs displayed elevated resting Ca levels, increased store Ca contents and facilitated store-operated Ca entry (SOCE) compared with RPMs. The intensified intracellular Ca stores were enriched with major luminal Ca-binding proteins inducibly expressed in TGPMs. The elevated resting Ca level was predominantly maintained by constitutive Ca influx, probably through the transient receptor potential (TRP) family members TRPP2, TRPM7 and TRPA1. These TRP family channels seemed to be largely activated in a manner dependent on phospholipase C activity, and together with Orai channels, contributed to SOCE. Moreover, Ca-dependent K channels efficiently facilitated SOCE by enhancing the Ca driving force in TGPMs. The consolidated cellular Ca handling described may underlie the specialized cell-physiological features of TGPMs, such as vital proliferation, active migration and avid phagocytosis.
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http://dx.doi.org/10.1016/j.ceca.2021.102381DOI Listing
February 2021

Decreased cardiac pacemaking and attenuated β-adrenergic response in TRIC-A knockout mice.

PLoS One 2020 21;15(12):e0244254. Epub 2020 Dec 21.

Department of Biological Chemistry, Kyoto University Graduate School and Faculty of Pharmaceutical Sciences, Kyoto, Japan.

Changes in intracellular calcium levels in the sinus node modulate cardiac pacemaking (the calcium clock). Trimeric intracellular cation (TRIC) channels are counterion channels on the surface of the sarcoplasmic reticulum and compensate for calcium release from ryanodine receptors, which play a major role in calcium-induced calcium release (CICR) and the calcium clock. TRIC channels are expected to affect the calcium clock in the sinus node. However, their physiological importance in cardiac rhythm formation remains unclear. We evaluated the importance of TRIC channels on cardiac pacemaking using TRIC-A-null (TRIC-A-/-) as well as TRIC-B+/-mice. Although systolic blood pressure (SBP) was not significantly different between wild-type (WT), TRIC-B+/-, and TRIC-A-/-mice, heart rate (HR) was significantly lower in TRIC-A-/-mice than other lines. Interestingly, HR and SBP showed a positive correlation in WT and TRIC-B+/-mice, while no such correlation was observed in TRIC-A-/-mice, suggesting modification of the blood pressure regulatory system in these mice. Isoproterenol (0.3 mg/kg) increased the HR in WT mice (98.8 ± 15.1 bpm), whereas a decreased response in HR was observed in TRIC-A-/-mice (23.8 ± 5.8 bpm), suggesting decreased sympathetic responses in TRIC-A-/-mice. Electrocardiography revealed unstable R-R intervals in TRIC-A-/-mice. Furthermore, TRIC-A-/-mice sometimes showed sinus pauses, suggesting a significant role of TRIC-A channels in cardiac pacemaking. In isolated atrium contraction or action potential recording, TRIC-A-/-mice showed decreased response to a β-adrenergic sympathetic nerve agonist (isoproterenol, 100 nM), indicating decreased sympathetic responses. In summary, TRIC-A-/-mice showed decreased cardiac pacemaking in the sinus node and attenuated responses to β-adrenergic stimulation, indicating the involvement of TRIC-A channels in cardiac rhythm formation and decreased sympathetic responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244254PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751866PMC
December 2020

TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle.

Circ Res 2020 02 6;126(4):417-435. Epub 2019 Dec 6.

From the Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University, Columbus (X. Zhou, K.H.P., P.-h.L., J.M.).

Rationale: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored.

Objective: In cardiomyocytes, spontaneous Ca waves, triggered by store overload-induced Ca release mediated by the RyR (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR-mediated Ca release machinery that directly modulates store overload-induced Ca release activity via CTT.

Methods And Results: We show that cardiomyocytes derived from the TRIC-A (TRIC-A knockout) mice display dysregulated Ca movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR. Modeling and docking studies reveal potential sites on RyR that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca oscillations. Ca measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca leak across the endoplasmic reticulum by directly targeting RyR to modulate store overload-induced Ca release. Moreover, synthetic CTT-A peptide facilitates RyR activity in lipid bilayer reconstitution system, enhances Ca sparks in permeabilized TRIC-A cardiomyocytes, and induces intracellular Ca release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates.

Conclusions: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR to modulate sarcoplasmic reticulum Ca handling in cardiac muscle.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035183PMC
February 2020

TRPM7 channels mediate spontaneous Ca fluctuations in growth plate chondrocytes that promote bone development.

Sci Signal 2019 04 9;12(576). Epub 2019 Apr 9.

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 615-8501, Japan.

During endochondral ossification of long bones, the proliferation and differentiation of chondrocytes cause them to be arranged into layered structures constituting the epiphyseal growth plate, where they secrete the cartilage matrix that is subsequently converted into trabecular bone. Ca signaling has been implicated in chondrogenesis in vitro. Through fluorometric imaging of bone slices from embryonic mice, we demonstrated that live growth plate chondrocytes generated small, cell-autonomous Ca fluctuations that were associated with weak and intermittent Ca influx. Several genes encoding Ca-permeable channels were expressed in growth plate chondrocytes, but only pharmacological inhibitors of transient receptor potential cation channel subfamily M member 7 (TRPM7) reduced the spontaneous Ca fluctuations. The TRPM7-mediated Ca influx was likely activated downstream of basal phospholipase C activity and was potentiated upon cell hyperpolarization induced by big-conductance Ca-dependent K channels. Bones from embryos in which was conditionally knocked out during ex vivo culture exhibited reduced outgrowth and displayed histological abnormalities accompanied by insufficient autophosphorylation of Ca/calmodulin-dependent protein kinase II (CaMKII) in the growth plate. The link between TRPM7-mediated Ca fluctuations and CaMKII-dependent chondrogenesis was further supported by experiments with chondrocyte-specific knockout mice. Thus, growth plate chondrocytes generate spontaneous, TRPM7-mediated Ca fluctuations that promote self-maturation and bone development.
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http://dx.doi.org/10.1126/scisignal.aaw4847DOI Listing
April 2019

Enhanced activity of multiple TRIC-B channels: an endoplasmic reticulum/sarcoplasmic reticulum mechanism to boost counterion currents.

J Physiol 2019 05 14;597(10):2691-2705. Epub 2019 Apr 14.

Department of Pharmacology, University of Oxford, Oxford, UK.

Key Points: There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single-channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K -channels from wild-type (WT) mice (where TRIC-A is the principal subtype) and from Tric-a knockout (KO) mice that only express TRIC-B. We find that lone SR K -channels from Tric-a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six-fold increase in activity when four channels are present in the bilayer. No evidence was found for a direct gating interaction between ryanodine receptor and SR K -channels in Tric-a KO SR, suggesting that TRIC-B-TRIC-B interactions are highly specific and may be important for meeting counterion requirements during excitation-contraction coupling in tissues where TRIC-A is sparse or absent.

Abstract: The trimeric intracellular cation channels, TRIC-A and TRIC-B, represent two subtypes of sarcoplasmic reticulum (SR) K -channel but their individual functional roles are unknown. We therefore compared the biophysical properties of SR K -channels derived from the skeletal muscle of wild-type (WT) or Tric-a knockout (KO) mice. Because TRIC-A is the major TRIC-subtype in skeletal muscle, WT SR will predominantly contain TRIC-A channels, whereas Tric-a KO SR will only contain TRIC-B channels. When lone SR K -channels were incorporated into bilayers, the open probability (Po) of channels from Tric-a KO mice was markedly lower than that of channels from WT mice; gating was characterized by shorter opening bursts and more frequent brief subconductance openings. However, unlike channels from WT mice, the Po of SR K -channels from Tric-a KO mice increased as increasing channel numbers were present in the bilayer, driving the channels into long sojourns in the fully open state. When co-incorporated into bilayers, ryanodine receptor channels did not directly affect the gating of SR K -channels, nor did the presence or absence of SR K -channels influence ryanodine receptor activity. We suggest that because of high expression levels in striated muscle, TRIC-A produces most of the counterion flux required during excitation-contraction coupling. TRIC-B, in contrast, is sparsely expressed in most cells and, although lone TRIC-B channels exhibit low Po, the high Po levels reached by multiple TRIC-B channels may provide a compensatory mechanism to rapidly restore K gradients and charge differences across the SR of tissues containing few TRIC-A channels.
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http://dx.doi.org/10.1113/JP277241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567852PMC
May 2019

Gm7325 is MyoD-dependently expressed in activated muscle satellite cells.

Biomed Res 2017 ;38(3):215-219

Graduate School of Pharmaceutical Sciences, Kyoto University.

The Gm7325 gene, bioinformatically identified in the mouse genome, encodes a small protein but has not been characterized until recently. Our gene expression analysis revealed that Gm7325 transcription is remarkably upregulated in injured skeletal muscle tissues. Activated satellite cells and immature myotubes were densely decorated with positive signals for Gm7325 mRNA in in situ hybridization analysis, while no obvious signals were observed in quiescent satellite cells and mature myofibers. In the 5'-flanking regions of mouse Gm7325 and its human homologue, conserved E-box motifs for helix-loop-helix transcription factors are repeatedly arranged around the putative promoter regions. Reporter gene assays suggested that MyoD, a master transcription factor for myogenesis, binds to the conserved E-box motifs to activate Gm7325 expression. Therefore, Gm7325, as a novel MyoD-target gene, is specifically induced in activated satellite cells, and may have an important role in skeletal myogenesis.
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http://dx.doi.org/10.2220/biomedres.38.215DOI Listing
March 2018

Dysregulated Zn homeostasis impairs cardiac type-2 ryanodine receptor and mitsugumin 23 functions, leading to sarcoplasmic reticulum Ca leakage.

J Biol Chem 2017 08 19;292(32):13361-13373. Epub 2017 Jun 19.

From the School of Medicine, University of St. Andrews, St. Andrews, KY16 9TF, Scotland, United Kingdom,

Aberrant Zn homeostasis is associated with dysregulated intracellular Ca release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca leakage through an uncharacterized mechanism. We sought to examine the role of Zn in shaping intracellular Ca release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca-permeable channels regulated by Zn Elevating the cytosolic Zn concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca leakage.
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http://dx.doi.org/10.1074/jbc.M117.781708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555195PMC
August 2017

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

J Physiol 2017 07 23;595(14):4769-4784. Epub 2017 May 23.

Department of Pharmacology, University of Oxford, Oxford, UK.

Key Points: The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric-a gene knockout (KO) alters the single-channel function of skeletal RyR (RyR1). We find that RyR1 from Tric-a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca , ATP, caffeine and luminal Ca . In the presence of Mg , ATP cannot effectively activate RyR1 from Tric-a KO mice. Additionally, RyR1 from Tric-a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β-adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca -release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca -release observed in skeletal muscle from Tric-a KO mice, further highlighting the importance of TRIC-A for normal physiological regulation of SR Ca -release in skeletal muscle.

Abstract: The type A trimeric intracellular cation channel (TRIC-A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric-a knockout (KO) mice is characterized by Ca overloaded and swollen SR and by changes in the properties of SR Ca release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric-a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage-clamp conditions. We find that RyR1 channels from Tric-a KO mice respond normally to cytosolic Ca , ATP, adenine, caffeine and to luminal Ca . However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg , ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC-A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC-A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K fluxes across the SR via TRIC-A.
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http://dx.doi.org/10.1113/JP273550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5509884PMC
July 2017

Optimizing conditions for calcium phosphate mediated transient transfection.

Saudi J Biol Sci 2017 Mar 11;24(3):622-629. Epub 2017 Feb 11.

College of Life Science, Shaanxi Normal University, Xi'an 710062, China.

Background: Calcium phosphate mediated transfection has been used for delivering DNA into mammalian cells in excess of 30 years due to its most low cost for introducing recombinant DNA into culture cells. However, multiple factors affecting the transfect efficiency are commonly recognized meanwhile for years, the low transfection efficiency of this approach on higher differentiated and non-tumor cells such as CHO and C2C12 limits its application on research.

Results: In this paper, we systematically evaluated the possible factors affecting the transfection rate of this approach. Two categories, calcium phosphate-DNA co-precipitation and on-cell treatments were set for optimization of plasmid DNA transfection into CHO and C2C12 cell-lines. Throughout experimentation of these categories such as buffer system, transfection media and time, glycerol shocking and so on, we optimized the best procedure to obtain the highest efficiency ultimately. During calcium phosphate DNA-precipitation, the transfection buffer is critical condition optimized with HBS at pH 7.10 ( = 0.013 compared to HEPES in CHO). In the transfection step, FBS is a necessary component in transfection DMEM for high efficiency ( = 0.0005 compared to DMEM alone), and high concentration of co-precipitated particles applied to cultured cells in combination with intermittent vortexing is also crucial to preserve the efficiency. For 6-well culture plates, 800 µl of co-precipitated particles (11.25 µg/mL of cDNA) in 1 well is the optimal ( = 0.007 compared to 200 µl). For the highest transfection efficiency, the most important condition is glycerol in shock treatment ( = 0.002 compared to no shock treatment in CHO, and  = 0.008 compared to no shock treatment in C2C12) after a 6 h incubation ( = 0.004 compared to 16 h in CHO, and  = 0.039 compared to 16 h in C2C12) on cultured cells.

Conclusions: Calcium phosphate mediated transfection is the most low-cost approach to introduce recombinant DNA into culture cells. However, the utility of this procedure is limited in highly-differentiated cells. Here we describe the specific HBS-buffered saline, PH, glycerol shock, vortex strength, transfection medium, and particle concentrations conditions necessary to optimize this transfection method in highly differentiated cells.
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http://dx.doi.org/10.1016/j.sjbs.2017.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372392PMC
March 2017

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

Sci Signal 2016 05 17;9(428):ra49. Epub 2016 May 17.

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.

The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.
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http://dx.doi.org/10.1126/scisignal.aad9055DOI Listing
May 2016

Junctophilin-4, a component of the endoplasmic reticulum-plasma membrane junctions, regulates Ca2+ dynamics in T cells.

Proc Natl Acad Sci U S A 2016 Mar 29;113(10):2762-7. Epub 2016 Feb 29.

Department of Physiology, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA 90095;

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.
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http://dx.doi.org/10.1073/pnas.1524229113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790987PMC
March 2016

TRIM72 modulates caveolar endocytosis in repair of lung cells.

Am J Physiol Lung Cell Mol Physiol 2016 Mar 4;310(5):L452-64. Epub 2015 Dec 4.

Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, Virginia; Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio; Division of Pulmonary, Critical Care, Allergy and Sleep Medicine, College of Medicine, The Ohio State University, Columbus, Ohio;

Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72's role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.
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http://dx.doi.org/10.1152/ajplung.00089.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773847PMC
March 2016

Subconductance gating and voltage sensitivity of sarcoplasmic reticulum K(+) channels: a modeling approach.

Biophys J 2015 Jul;109(2):265-76

Department of Pharmacology, University of Oxford, Oxford, United Kingdom. Electronic address:

Sarcoplasmic reticulum (SR) K(+) channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K(+) channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K(+) channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K(+) channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K(+) channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (≤0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K(+) flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.
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http://dx.doi.org/10.1016/j.bpj.2015.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623209PMC
July 2015

Comprehensive behavioral analysis of voltage-gated calcium channel beta-anchoring and -regulatory protein knockout mice.

Front Behav Neurosci 2015 16;9:141. Epub 2015 Jun 16.

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University Kyoto, Japan.

Calcium (Ca(2+)) influx through voltage-gated Ca(2+) channels (VGCCs) induces numerous intracellular events such as neuronal excitability, neurotransmitter release, synaptic plasticity, and gene regulation. It has been shown that genes related to Ca(2+) signaling, such as the CACNA1C, CACNB2, and CACNA1I genes that encode VGCC subunits, are associated with schizophrenia and other psychiatric disorders. Recently, VGCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of VGCC activity via the interaction of VGCC β subunits. To examine the role of the BARP in higher brain functions, we generated BARP knockout (KO) mice and conducted a comprehensive battery of behavioral tests. BARP KO mice exhibited greatly reduced locomotor activity, as evidenced by decreased vertical activity, stereotypic counts in the open field test, and activity level in the home cage, and longer latency to complete a session in spontaneous T-maze alteration test, which reached "study-wide significance." Acoustic startle response was also reduced in the mutants. Interestingly, they showed multiple behavioral phenotypes that are seemingly opposite to those seen in the mouse models of schizophrenia and its related disorders, including increased working memory, flexibility, prepulse inhibition, and social interaction, and decreased locomotor activity, though many of these phenotypes are statistically weak and require further replications. These results demonstrate that BARP is involved in the regulation of locomotor activity and, possibly, emotionality. The possibility was also suggested that BARP KO mice may serve as a unique tool for investigating the pathogenesis/pathophysiology of schizophrenia and related disorders. Further evaluation of the molecular and physiological phenotypes of the mutant mice would provide new insights into the role of BARP in higher brain functions.
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http://dx.doi.org/10.3389/fnbeh.2015.00141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468383PMC
July 2015

Mitsugumin 56 (hedgehog acyltransferase-like) is a sarcoplasmic reticulum-resident protein essential for postnatal muscle maturation.

FEBS Lett 2015 Apr 1;589(10):1095-104. Epub 2015 Apr 1.

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan; Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kyoto 606-8501, Japan. Electronic address:

Mitsugumin 56 (MG56), also known as the membrane-bound O-acyl-transferase family member hedgehog acyltransferase-like, was identified as a new sarcoplasmic reticulum component in striated muscle. Mg56-knockout mice grew normally for a week after birth, but shortly thereafter exhibited a suckling defect and died under starvation conditions. In the knockout skeletal muscle, regular contractile features were largely preserved, but sarcoplasmic reticulum elements swelled and further developed enormous vacuoles. In parallel, the unfolded protein response was severely activated in the knockout muscle, and presumably disrupted muscle development leading to the suckling failure. Therefore, MG56 seems essential for postnatal skeletal muscle maturation.
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http://dx.doi.org/10.1016/j.febslet.2015.03.028DOI Listing
April 2015

TRIM72 is required for effective repair of alveolar epithelial cell wounding.

Am J Physiol Lung Cell Mol Physiol 2014 Sep 8;307(6):L449-59. Epub 2014 Aug 8.

Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio; Division of Pulmonary, Critical Care, Allergy and Sleep Medicine, College of Medicine, The Ohio State University, Columbus, Ohio

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.
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http://dx.doi.org/10.1152/ajplung.00172.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166787PMC
September 2014

Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.

Nat Commun 2014 Jul 18;5:4387. Epub 2014 Jul 18.

Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210.

Injury to lung epithelial cells has a role in multiple lung diseases. We previously identified mitsugumin 53 (MG53) as a component of the cell membrane repair machinery in striated muscle cells. Here we show that MG53 also has a physiological role in the lung and may be used as a treatment in animal models of acute lung injury. Mice lacking MG53 show increased susceptibility to ischaemia-reperfusion and overventilation-induced injury to the lung when compared with wild-type mice. Extracellular application of recombinant human MG53 (rhMG53) protein protects cultured lung epithelial cells against anoxia/reoxygenation-induced injuries. Intravenous delivery or inhalation of rhMG53 reduces symptoms in rodent models of acute lung injury and emphysema. Repetitive administration of rhMG53 improves pulmonary structure associated with chronic lung injury in mice. Our data indicate a physiological function for MG53 in the lung and suggest that targeting membrane repair may be an effective means for treatment or prevention of lung diseases.
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http://dx.doi.org/10.1038/ncomms5387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109002PMC
July 2014

Aberrant behavioral sensitization by methamphetamine in junctophilin-deficient mice.

Mol Neurobiol 2015 Apr 22;51(2):533-42. Epub 2014 May 22.

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-Aoba, Aoba-ku, Sendai, Miyagi, 980-8578, Japan,

Junctophilins (JPs) expressed in the endoplasmic/sarcoplasmic reticulum (ER/SR) interact with the plasma membrane, thereby constructing junctional membrane complexes (JMC). We here reported that double-knockout mice lacking both JP3 and JP4 (JP-DKO mice) exhibit aberrant synaptic plasticity in the corticostriatal circuits and irregular methamphetamine (METH)-induced behavioral sensitization when METH (1.0 mg/kg) was administrated six consecutive days and assessed the striatal glutamatergic population spike (PS) by stimulation of cortical white matter. When we assessed the striatal PS by stimulation of cortical white matter, the long-term depression (LTD) was observed in JP-DKO mouse striatum similar to that in control (JP-double hetero mice (JP-DHE mice)). Importantly, LTD converted to long-term potentiation (LTP) following chronic METH treatment concomitant with behavioral sensitization in JP-DHE mice. LTD in JP-DKO mice, however failed to convert to LTP with lacks of behavioral sensitization. LTP impairment in JP-DKO mice was restored by pretreatment with FK506, calcineurin (CaN) inhibitor, but not with apamin, SK channel inhibitor. In immunoblotting analyses, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation was significantly increased following METH treatment in the striatum of JP-DHE mice. However, CaMKII autophosphorylation did not changed by METH treatment in the striatum of JP-DKO mouse. The increased CaMKII autophosphorylation was closely associated with elevated CaN activity in JP-DKO mice. The lack of increased CaMKII activity in JP-DKO mice was correlated with the impaired METH-induced behavioral sensitization. Thus, elevated CaN and aberrant CaMKII activities in the striatum of JP-DKO mice likely accounts for lack of METH-induced behavioral sensitization.
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http://dx.doi.org/10.1007/s12035-014-8737-2DOI Listing
April 2015

PRDM14 promotes active DNA demethylation through the ten-eleven translocation (TET)-mediated base excision repair pathway in embryonic stem cells.

Development 2014 Jan 11;141(2):269-80. Epub 2013 Dec 11.

Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.

Ten-eleven translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised and repaired by the base excision repair (BER) pathway, implicating 5mC oxidation in active DNA demethylation. Genome-wide DNA methylation is erased in the transition from metastable states to the ground state of embryonic stem cells (ESCs) and in migrating primordial germ cells (PGCs), although some resistant regions become demethylated only in gonadal PGCs. Understanding the mechanisms underlying global hypomethylation in naive ESCs and developing PGCs will be useful for realizing cellular pluripotency and totipotency. In this study, we found that PRDM14, the PR domain-containing transcriptional regulator, accelerates the TET-BER cycle, resulting in the promotion of active DNA demethylation in ESCs. Induction of Prdm14 expression transiently elevated 5hmC, followed by the reduction of 5mC at pluripotency-associated genes, germline-specific genes and imprinted loci, but not across the entire genome, which resembles the second wave of DNA demethylation observed in gonadal PGCs. PRDM14 physically interacts with TET1 and TET2 and enhances the recruitment of TET1 and TET2 at target loci. Knockdown of TET1 and TET2 impaired transcriptional regulation and DNA demethylation by PRDM14. The repression of the BER pathway by administration of pharmacological inhibitors of APE1 and PARP1 and the knockdown of thymine DNA glycosylase (TDG) also impaired DNA demethylation by PRDM14. Furthermore, DNA demethylation induced by PRDM14 takes place normally in the presence of aphidicolin, which is an inhibitor of G1/S progression. Together, our analysis provides mechanistic insight into DNA demethylation in naive pluripotent stem cells and developing PGCs.
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http://dx.doi.org/10.1242/dev.099622DOI Listing
January 2014

TRIC-B channels display labile gating: evidence from the TRIC-A knockout mouse model.

Pflugers Arch 2013 Aug 7;465(8):1135-48. Epub 2013 Mar 7.

School of Physiology & Pharmacology, NSQI and Bristol Heart Institute, University of Bristol, Bristol, BS8 1TD, UK.

Sarcoplasmic/endoplasmic reticulum (SR) and nuclear membranes contain two related cation channels named TRIC-A and TRIC-B. In many tissues, both subtypes are co-expressed, making it impossible to distinguish the distinct single-channel properties of each subtype. We therefore incorporated skeletal muscle SR vesicles derived from Tric-a-knockout mice into bilayers in order to characterise the biophysical properties of native TRIC-B without possible misclassification of the channels as TRIC-A, and without potential distortion of functional properties by detergent purification protocols. The native TRIC-B channels were ideally selective for cations. In symmetrical 210 mM K(+), the maximum (full) open channel level (199 pS) was equivalent to that observed when wild-type SR vesicles were incorporated into bilayers. Analysis of TRIC-B gating revealed complex and variable behaviour. Four main sub-conductance levels were observed at approximately 80 % (161 pS), 60 % (123 pS), 46 % (93 pS), and 30 % (60 pS) of the full open state. Seventy-five percent of the channels were voltage sensitive with Po being markedly reduced at negative holding potentials. The frequent, rapid transitions between TRIC-B sub-conductance states prevented development of reliable gating models using conventional single-channel analysis. Instead, we used mean-variance plots to highlight key features of TRIC-B gating in a more accurate and visually useful manner. Our study provides the first biophysical characterisation of native TRIC-B channels and indicates that this channel would be suited to provide counter current in response to Ca(2+) release from the SR. Further experiments are required to distinguish the distinct functional properties of TRIC-A and TRIC-B and understand their individual but complementary physiological roles.
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http://dx.doi.org/10.1007/s00424-013-1251-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732801PMC
August 2013

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice.

Nat Commun 2013 ;4:1541

Department of Experimental and Diagnostic Medicine, General Pathology section, University of Ferrara, Via Borsari 46, Ferrara 44121, Italy.

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.
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http://dx.doi.org/10.1038/ncomms2496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856057PMC
June 2013

Essential roles of the histone methyltransferase ESET in the epigenetic control of neural progenitor cells during development.

Development 2012 Oct;139(20):3806-16

Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.

In the developing brain, neural progenitor cells switch differentiation competency by changing gene expression profiles that are governed partly by epigenetic control, such as histone modification, although the precise mechanism is unknown. Here we found that ESET (Setdb1), a histone H3 Lys9 (H3K9) methyltransferase, is highly expressed at early stages of mouse brain development but downregulated over time, and that ablation of ESET leads to decreased H3K9 trimethylation and the misregulation of genes, resulting in severe brain defects and early lethality. In the mutant brain, endogenous retrotransposons were derepressed and non-neural gene expression was activated. Furthermore, early neurogenesis was severely impaired, whereas astrocyte formation was enhanced. We conclude that there is an epigenetic role of ESET in the temporal and tissue-specific gene expression that results in proper control of brain development.
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http://dx.doi.org/10.1242/dev.082198DOI Listing
October 2012

TRIM50 protein regulates vesicular trafficking for acid secretion in gastric parietal cells.

J Biol Chem 2012 Sep 7;287(40):33523-32. Epub 2012 Aug 7.

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.
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http://dx.doi.org/10.1074/jbc.M112.370551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460453PMC
September 2012

Endogenously determined restriction of food intake overcomes excitation-contraction uncoupling in JP45KO mice with aging.

Exp Gerontol 2012 Apr 25;47(4):304-16. Epub 2012 Jan 25.

Department of Internal Medicine, Section on Gerontology and Geriatric Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, United States.

The decline in muscular strength with age is disproportionate to the loss in total muscle mass that causes it. Knocking out JP45, an integral protein of the junctional face membrane of the skeletal muscle sarcoplasmic reticulum (SR), results in decreased expression of the voltage-gated Ca(2+) channel, Ca(v)1.1; excitation-contraction uncoupling (ECU); and loss of muscle force (Delbono et al., 2007). Here, we show that Ca(v)1.1 expression, charge movement, SR Ca(2+) release, in vitro contractile force, and sustained forced running remain stable in male JP45KO mice at 12 and 18 months. They also exhibit the level of ECU reported for 3-4-month mice (Delbono et al., 2007). No further decline at later ages was recorded. Preserved ECC was not related to increased expression of any protein that directly or indirectly interacts with JP45 at the triad junction. However, maintained muscle force and physical performance were associated with ablation of JP45 expression in the brain, spontaneous and significantly diminished food intake and less tendency toward obesity when exposed to a high-fat diet compared to WT. We propose that (1) endogenously generated restriction in food intake overcomes the deleterious effects of JP45 ablation on ECC and skeletal muscle force mainly through downregulation of neuropeptide-Y expression in the hypothalamic arcuate nucleus; and (2) the JP45KO mouse constitutes an invaluable model to examine the mechanisms controlling food intake as well as skeletal muscle function with aging.
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http://dx.doi.org/10.1016/j.exger.2012.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307889PMC
April 2012

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance.

Cell Metab 2011 Aug;14(2):231-41

Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.
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http://dx.doi.org/10.1016/j.cmet.2011.05.011DOI Listing
August 2011

Mitsugumin 23 forms a massive bowl-shaped assembly and cation-conducting channel.

Biochemistry 2011 Apr 7;50(13):2623-32. Epub 2011 Mar 7.

School of Physiology and Pharmacology, Bristol Heart Institute and Centre for Nanoscience and Quantum Information, University of Bristol, Bristol, United Kingdom.

Mitsugumin 23 (MG23) is a 23 kDa transmembrane protein localized to the sarcoplasmic/endoplasmic reticulum and nuclear membranes in a wide variety of cells. Although the characteristics imply the participation in a fundamental function in intracellular membrane systems, the physiological role of MG23 is unknown. Here we report the biochemical and biophysical characterization of MG23. Hydropathicity profile and limited proteolytic analysis proposed three transmembrane segments in the MG23 primary structure. Chemical cross-linking analysis suggested a homo-oligomeric assembly of MG23. Ultrastructural observations detected a large symmetrical particle as the predominant component and a small asymmetric assembly as the second major component in highly purified MG23 preparations. Single-particle three-dimensional reconstruction revealed that MG23 forms a large bowl-shaped complex equipped with a putative central pore, which is considered an assembly of the small asymmetric subunit. After reconstitution into planar phospholipid bilayers, purified MG23 behaved as a voltage-dependent, cation-conducting channel, permeable to both K(+) and Ca(2+). A feature of MG23 gating was that multiple channels always appeared to be gating together in the bilayer. Our observations suggest that the bowl-shaped MG23 can transiently assemble and disassemble. These building transitions may underlie the unusual channel gating behavior of MG23 and allow rapid cationic flux across intracellular membrane systems.
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http://dx.doi.org/10.1021/bi1019447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065873PMC
April 2011

Ca2+ overload and sarcoplasmic reticulum instability in tric-a null skeletal muscle.

J Biol Chem 2010 Nov 21;285(48):37370-6. Epub 2010 Sep 21.

Department of Physiology and Biophysics, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

The sarcoplasmic reticulum (SR) of skeletal muscle contains K(+), Cl(-), and H(+) channels may facilitate charge neutralization during Ca(2+) release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca(2+) release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a(-/-) skeletal muscle showed Ca(2+) overload inside the SR with frequent formation of Ca(2+) deposits compared with the wild type muscle. This elevated SR Ca(2+) pool in the tric-a(-/-) muscle could be released by caffeine, whereas the elemental Ca(2+) release events, e.g. osmotic stress-induced Ca(2+) spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of "alternan" behavior with isolated tric-a(-/-) skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca(2+ ATPase) function could lead to aggravation of the stress-induced alternans in the tric-a(-/-) muscle. Our data suggests that absence of TRIC-A may lead to Ca(2+) overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca(2+) movement across the SR membrane. The observed alternan behavior with the tric-a(-/-) muscle may reflect a skeletal muscle version of store overload-induced Ca(2+) release that has been reported in the cardiac muscle under stress conditions.
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http://dx.doi.org/10.1074/jbc.M110.170084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988342PMC
November 2010

Charade of the SR K+-channel: two ion-channels, TRIC-A and TRIC-B, masquerade as a single K+-channel.

Biophys J 2010 Jul;99(2):417-26

Department of Physiology and Pharmacology and Bristol Heart Institute, University of Bristol, Bristol, United Kingdom.

The presence of a sarcoplasmic reticulum (SR) K+-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K+-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K+ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K+-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K+-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca2+ and inhibited by luminal Ca2+. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.
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http://dx.doi.org/10.1016/j.bpj.2010.04.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2905108PMC
July 2010

MG53 constitutes a primary determinant of cardiac ischemic preconditioning.

Circulation 2010 Jun 1;121(23):2565-74. Epub 2010 Jun 1.

Institute of Molecular Medicine, Peking University, Beijing, China.

Background: Ischemic heart disease is the greatest cause of death in Western countries. The deleterious effects of cardiac ischemia are ameliorated by ischemic preconditioning (IPC), in which transient ischemia protects against subsequent severe ischemia/reperfusion injury. IPC activates multiple signaling pathways, including the reperfusion injury salvage kinase pathway (mainly PI3K-Akt-glycogen synthase kinase-3beta [GSK3beta] and ERK1/2) and the survivor activating factor enhancement pathway involving activation of the JAK-STAT3 axis. Nevertheless, the fundamental mechanism underlying IPC is poorly understood.

Methods And Results: In the present study, we define MG53, a muscle-specific TRIM-family protein, as a crucial component of cardiac IPC machinery. Ischemia/reperfusion or hypoxia/oxidative stress applied to perfused mouse hearts or neonatal rat cardiomyocytes, respectively, causes downregulation of MG53, and IPC can prevent ischemia/reperfusion-induced decrease in MG53 expression. MG53 deficiency increases myocardial vulnerability to ischemia/reperfusion injury and abolishes IPC protection. Overexpression of MG53 attenuates whereas knockdown of MG53 enhances hypoxia- and H(2)O(2)-induced cardiomyocyte death. The cardiac protective effects of MG53 are attributable to MG53-dependent interaction of caveolin-3 with phosphatidylinositol 3 kinase and subsequent activation of the reperfusion injury salvage kinase pathway without altering the survivor activating factor enhancement pathway.

Conclusions: These results establish MG53 as a primary component of the cardiac IPC response, thus identifying a potentially important novel therapeutic target for the treatment of ischemic heart disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.110.954628DOI Listing
June 2010

Facilitation of DNA damage-induced apoptosis by endoplasmic reticulum protein mitsugumin23.

Biochem Biophys Res Commun 2010 Feb 12;392(2):196-200. Epub 2010 Jan 12.

Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.

The endoplasmic reticulum (ER) emanates context-dependent signals, thereby mediating cellular response to a variety of stresses. However, the underlying molecular mechanisms have been enigmatic. To better understand the signaling capacity of the ER, we focused on roles played by mitsugumin23 (MG23), a protein residing predominantly in this organelle. Overexpression of MG23 in human embryonic kidney 293T cells specifically enhanced apoptosis triggered by etoposide, a DNA-damaging anti-cancer drug. Conversely, genetic deletion of MG23 reduced susceptibility of thymocytes to DNA damage-induced apoptosis, which was demonstrated by whole-body irradiation experiments. In this setting, induction of the tumor-suppressor gene p53 was attenuated in MG23-knockout thymocytes as compared with their wild-type counterparts, consistent with the elevated radioresistance. It is therefore suggested that MG23 is an essential component of ER-generated lethal signals provoked upon DNA damage, specifying cell fate under pathophysiological conditions.
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http://dx.doi.org/10.1016/j.bbrc.2010.01.013DOI Listing
February 2010